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PublicatienummerCN103937871 A
PublicatietypeAanvraag
AanvraagnummerCN 201310025221
Publicatiedatum23 juli 2014
Aanvraagdatum23 jan 2013
Prioriteitsdatum23 jan 2013
Publicatienummer201310025221.X, CN 103937871 A, CN 103937871A, CN 201310025221, CN-A-103937871, CN103937871 A, CN103937871A, CN201310025221, CN201310025221.X
Uitvinders高勇, 李砚东
Aanvrager上海市东方医院
Citatie exporterenBiBTeX, EndNote, RefMan
Externe links:  SIPO, Espacenet
Application of SRRP35 gene and expression product thereof to cancer diagnosis and treatment
CN 103937871 A
Samenvatting
The invention discloses application of SRRP35 gene and an expression product thereof to prepare products for cancer diagnosis and treatment. SRRP35 gene and the expression product can be used a specific marker gene for diagnosing cancers especially liver cancer; and SRRP35 gene and the expression product also can be used as a target gene for preparing medicines for treating cancers especially liver cancer, and help to provide a new cancer treatment approach.
Claims(10)  vertaald uit het Chinees
1.一种SRRP35基因或SRRP35蛋白的用途,其特征在于,用于制备检测癌症的试剂或试剂盒。 A SRRP35 SRRP35 gene or protein use, characterized in that the detection of cancer for the preparation of reagents or kits.
2.如权利要求1所述的用途,其特征在于,所述的试剂盒包括:对SRRP35蛋白或mRNA进行定量检测的试剂以及相应的标签或说明书。 2. The use as claimed in claim 1, characterized in that, said kit comprising: SRRP35 quantifying mRNA or protein detection reagent and the corresponding label or instructions.
3.如权利要求1所述的用途,其特征在于,所述的试剂包括SRRP35特异性引物、特异性抗体、探针和/或芯片;和/或所述的癌症包括肝癌。 Use according to claim, characterized in that said reagent comprises SRRP35 specific primers, specific antibodies, probes, and / or chips; cancer and / or comprises liver.
4.一种用于检测癌症的诊断试剂盒,其特征在于,所述的试剂盒含有一容器,所述容器中含有检测SRRP35蛋白或mRNA的检测试剂;以及标签或说明书,所述标签或说明书注明所述试剂盒用于检测癌症。 4. A method for detecting a cancer diagnostic kit, wherein said kit comprising a container, said container containing protein or mRNA is detected SRRP35 detection reagent; and a label or instructions, the label or instructions indicate the kit for detecting cancer.
5.如权利要求4所述的试剂盒,其特征在于,所述的标签或说明书中注明以下内容: 当检测对象的SRRP35相对β -肌动蛋白的mRNA表达量与癌旁组织的SRRP35相对β -肌动蛋白的mRNA表达量之比≤1,则提示该检测对象患癌症的几率高于普通人群。 5. The kit according to claim 4, wherein said label or instructions indicate the following: When SRRP35 a detected object β - actin mRNA expression and adjacent tissues of SRRP35 relative β - actin mRNA expression level of the ratio of ≤1, then the probability of the prompt detection target cancer is higher than the general population.
6.一种SRRP35蛋白、SRRP35基因或其激动剂的用途,其特征在于,被用于制备抑制癌细胞生长或增殖的药物,或用于制备治疗癌症的药物。 A SRRP35 protein, SRRP35 gene or the use of an agonist, wherein the preparation is used to inhibit cancer cell growth or proliferation medicament or preparation of a medicament for the treatment of cancer.
7.—种体外非治疗性的抑制癌细胞生长或增殖的方法,其特征在于,包括步骤:在SRRP35蛋白或其激动剂存在下,培养癌细胞,从而抑制癌细胞生长或增殖。 7.- species in vitro method of non-therapeutic inhibition of cancer cell growth or proliferation, characterized by comprising the steps of: in the presence of an agonist or SRRP35 protein, cultured cancer cells, thereby inhibiting cancer cell growth or proliferation.
8.一种筛选治疗癌症的候选化合物的方法,其特征在于,所述方法包括步骤: (a)测试组中,在细胞的培养体系中添加测试化合物,并观察所述测试组的细胞中SRRP35的表达量和/或活性;在对照组中,在相同细胞的培养体系中不添加测试化合物,并观察对照组的所述细胞中SRRP35的表达量和/或活性; 其中,如果测试组中细胞的SRRP35的表达量和/或活性大于对照组,就表明该测试化合物是对SRRP35的表达和/或活性有促进作用的治疗癌症的候选化合物。 8. A method of screening a candidate compound for the treatment of cancer, characterized in that, said method comprising the steps of: (a) the test group, the test compound is added in the cell culture system, and the cells were observed in the test group SRRP35 the expression and / or activity; in the control group, in the same cell culture system does not add the test compound, the cells were observed in the control group SRRP35 expression and / or activity; wherein if the test group cells The expression of SRRP35 and / or activity than the control group, it indicates that the test compound is an expression of SRRP35 and / or activity of promoting a candidate compound for treating cancer.
9.如权利要求8所述的方法,其特征在于,所述方法还包括步骤: (b)对于步骤(a)中获得的候选化合物,进一步测试其对癌细胞生长或增殖的抑制作用。 9. The method according to claim 8, characterized in that the method further comprises the step of: (b) a candidate compound for step (a) obtained, further testing their inhibition of cell growth or proliferation.
10.如权利要求9所述的方法,其特征在于,所述步骤(b)中包括步骤:测试组中,癌细胞的培养体系中添加测试化合物,并观察癌细胞的数量和/或生长情况;在对照组中,在癌细胞的培养体系中不添加测试化合物,并观察癌细胞的数量和/或生长情况;其中,如果测试组中癌细胞的数量或生长速度小于对照组,就表明该测试化合物是对癌细胞的生长或增殖有抑制作用的治疗癌症的候选化合物。 10. The method according to claim 9, wherein said step (b) includes the steps of: test group, in the culture system of cancer cells to add the test compound, and the observed number of cancer cells and / or growth ; in the control group, the cancer cells in culture system does not add the test compound, and the number of observations and / or growth of cancer cells; wherein if the number of cancer cells in the test group or the growth rate than the control group, it indicates that the the test compound is growth of cancer cells or inhibit the proliferation of candidate compounds for the treatment of cancer.
Beschrijving  vertaald uit het Chinees

SRRP35基因和表达产物在癌症诊断与治疗中的应用 SRRP35 gene expression products and applications in cancer diagnosis and treatment

技术领域 Technical Field

[0001] 本发明涉及肿瘤学领域。 [0001] The present invention relates to the field of oncology. 更具体地,本发明涉及SRRP35基因和表达产物在癌症检测方面的应用,尤其是在肝癌的检测的应用。 More particularly, the present invention relates to SRRP35 gene expression products and applications in cancer detection, especially in the detection of liver cancer applications. 本发明还涉及SRRP35基因、蛋白及其激动剂在癌症治疗中的应用。 The present invention also relates to SRRP35 gene, protein and its agonist in cancer treatment applications.

背景技术 Background

[0002] 肝细胞癌(hepatocellularcarcinoma, HCC)是我国常见的恶性肿瘤,其死亡率位居第二。 [0002] carcinoma (hepatocellularcarcinoma, HCC) is the common malignant tumor, the mortality rate ranked second. 肝癌的诊断,尤其是早期诊断,是临床诊疗和预后的关键。 Liver cancer diagnosis, especially early diagnosis, clinical diagnosis and prognosis of the key. 可用作原发性肝癌标志物的生物分子,通常认为有以下四类:癌胚和糖蛋白抗原;酶和同工酶;细胞因子;基因。 It can be used as primary liver cancer marker of biological molecules, generally considered the following four categories: carcinoembryonic antigen and glycoproteins; enzyme and isoenzyme; cytokines; genes. 在全球范围内,对肝癌的定性诊断以检测血清AFP (甲胎蛋白)为主,但AFP的敏感性(40%~65%)和特异性(76%~96%)均不令人满意。 Worldwide, diagnosis of liver cancer in serum AFP (AFP) mainly, but AFP sensitivity (40% to 65%) and specificity (76% to 96%) are not satisfactory.

[0003] 此外,肝癌的治疗在过去20年里取得了很大的进展,出现了一些局部非手术治疗方案,但是,由于目前肝癌的化疗药物基本沿用其他肿瘤的治疗药物,针对性较差,因此,总体疗效不佳。 [0003] In addition, the treatment of liver cancer in the past 20 years has made great progress, there are some local non-surgical treatment options, however, due to the current liver cancer chemotherapy drugs basically follows other cancer treatment drugs, targeted poor, Thus, the overall effect is poor. [0004] 因此,本领域迫切需要开发可用于肝癌诊断的相关蛋白,为了有效地抑制肝癌细胞的生长,本领域迫切需要开发可用于抑制肝癌细胞生长的药物,以提高化疗的特异性和有效性。 [0004] Thus, there is an urgent need to develop related proteins can be used for diagnosis of liver cancer, in order to effectively inhibit the growth of liver cancer cells, there is an urgent need to develop drugs that can be used to inhibit the growth of liver cancer cells, in order to improve the specificity and effectiveness of chemotherapy .

发明内容 DISCLOSURE

[0005] 本发明公开了一种人SRRP35基因及其表达产物的应用,用于制备癌症尤其是肝癌的诊断及治疗产品。 [0005] The present invention discloses a human gene and its expression products SRRP35 application for the preparation of cancer, especially liver cancer diagnostic and therapeutic products.

[0006] 本发明的第一方面提供了一种SRRP35基因或SRRP35蛋白的用途,用于制备检测癌症的试剂或试剂盒; [0006] The first aspect of the present invention there is provided a SRRP35 SRRP35 gene or protein thereof, for the detection of cancer of the preparation of reagents or kits;

[0007] 在另一优选例中,所述的癌症是肝癌。 [0007] In another preferred embodiment, the cancer is liver cancer.

[0008] 在另一优选例中,所述的试剂盒包括:对SRRP35蛋白或mRNA进行定量检测的试剂以及相应的标签或说明书。 [0008] In another preferred embodiment, the kit includes: SRRP35 protein or mRNA quantitative detection reagent and the corresponding label or instructions.

[0009] 在另一优选例中,所述的试剂包括SRRP35特异性引物、特异性抗体、探针和/或芯片。 [0009] In another preferred embodiment, the reagent comprises SRRP35 specific primers, specific antibodies, probes, and / or chips.

[0010] 在另一优选例中,上述的试剂包括检测用芯片,包括核酸芯片和蛋白质芯片。 [0010] In another preferred embodiment, said reagent comprising detecting chip, the chip comprises a nucleic acid and protein chips.

[0011] 在另一优选例中,所述的核酸芯片包括基片和点样在基片上的癌症相关基因的特异性寡核苷酸探针,所述的癌症相关基因的特异性寡核苷酸探针包括与SRRP35基因或mRNA特异性结合的探针。 [0011] In another preferred embodiment, the nucleic acid chip-specific oligonucleotide probes comprising a substrate and spotting cancer-related genes in the substrate, said specific oligonucleotide cancer-related genes SRRP35 acid probes include probes that specifically binds to a gene or mRNA.

[0012] 在另一优选例中,所述的蛋白质芯片包括基片和点样在基片上的癌症相关蛋白的特异性抗体,所述的癌症相关蛋白的特异性抗体包括抗SRRP35蛋白的特异性抗体。 [0012] In another preferred embodiment, the protein chip comprising a substrate and spotted on a substrate cancer-associated protein-specific antibodies, the cancer-related protein-specific antibody proteins include specific anti SRRP35 antibodies.

[0013] 在另一优选例中,所述的SRRP35蛋白包括融合蛋白和非融合蛋白。 [0013] In another preferred embodiment, it said SRRP35 proteins include fusion proteins and non-fusion proteins.

[0014] 本发明的第二方面,提供了一种用于检测癌症的诊断试剂盒,所述的试剂盒含有一容器,所述容器中含有检测SRRP35蛋白或mRNA的检测试剂;以及标签或说明书,所述标签或说明书注明所述试剂盒用于检测癌症。 [0014] The second aspect of the present invention, there is provided a method for detecting a cancer diagnostic kit, said kit comprising a container, said container containing protein or mRNA is detected SRRP35 detection reagent; and a label or instructions , the label or instructions for the kit indicate detection of cancer.

[0015] 在另一优选例中,所述的标签或说明书中注明以下内容: [0015] In another preferred embodiment, the label or instructions indicate the following:

[0016] 当检测对象的SRRP35相对β -肌动蛋白的mRNA表达量与癌旁组织的SRRP35相对肌动蛋白的mRNA表达量之比≤ 1,则提示该检测对象患癌症的几率高于普通人群。 [0016] When SRRP35 a detected object β - actin mRNA expression and adjacent tissues of SRRP35 relatively actin mRNA expression ratio of ≤ 1, the system prompts the detection object risk of cancer is higher than the general population .

[0017] 在另一优选例中,所述的检测试剂包括:特异性引物、特异性抗体、探针和/或芯片; [0017] In another preferred embodiment, the detection reagent comprising: specific primers, specific antibodies, probes, and / or chip;

[0018] 在另一优选例中,所述的试剂盒用于检测人肿瘤组织样品或血液样品; [0018] In another preferred embodiment, the kit for detecting human tumor tissue sample or a blood sample;

[0019] 在另一优选例中,所述的肿瘤组织样品为肝癌样品。 [0019] In another preferred embodiment, said tumor tissue sample is a sample of liver cancer.

[0020] 本发明的第三方面,提供了一种SRRP35蛋白、SRRP35基因或其激动剂的用途,用于制备抑制癌细胞生长或增殖的药物,或用于制备治疗癌症的药物。 [0020] The third aspect of the invention, there is provided a SRRP35 protein, SRRP35 gene or an agonist thereof, for the preparation inhibit cancer cell growth or proliferation of drugs, or the preparation of a medicament for the treatment of cancer.

[0021] 本发明的第四方面,提供了一种体外非治疗性的抑制癌细胞生长或增殖的方法,包括步骤:在SRRP35蛋白或其激动剂存在下,培养癌细胞,从而抑制癌细胞生长或增殖。 [0021] A fourth aspect of the present invention, there is provided a method of non-therapeutic in vitro inhibition of growth or proliferation of cancer cells, comprising the steps of: in the presence of an agonist or SRRP35 protein, cultured cancer cells, thereby inhibiting cancer cell growth or proliferation.

[0022] 在另一优选例中,所述的方法包括向癌细胞的培养体系中添加SRRP35激动剂,从而抑制癌细胞生长或增殖。 [0022] In another preferred embodiment, said method comprises adding to the culture system SRRP35 agonist in cancer cells, thereby inhibiting cancer cell growth or proliferation.

[0023] 在另一优选例中,所述的癌细胞是肝癌细胞。 [0023] In another preferred embodiment, the cancer is hepatoma cells.

[0024] 本发明的第五方面,提供了一种筛选治疗癌症的候选化合物的方法,包括步骤: The fifth aspect of the [0024] present invention, there is provided a method of treating cancer screening a candidate compound, comprising the steps of:

[0025] (a)测试组中,在细胞的培养体系中添加测试化合物,并观察所述测试组的细胞中SRRP35的表达量和/或活性;在对照组中,在相同细胞的培养体系中不添加测试化合物,并观察对照组的所述细胞中SRRP35的表达量和/或活性; [0025] (a) the test group, add a test compound in culture system cells, and the cells were observed in the test group SRRP35 expression and / or activity; in the control group, in the same culture system cells Do not add the test compound, the cells were observed in the control group SRRP35 expression and / or activity;

[0026] 其中,如果测试组中细胞的SRRP35的表达量和/或活性大于对照组,就表明该测试化合物是对SRRP35的表达和/或活性有促进作用的治疗癌症的候选化合物。 [0026] where, if the expression of cell SRRP35 test group and / or activity than the control group, it indicates that the test compound is SRRP35 expression and / or activity of promoting a candidate compound for treating cancer.

[0027] 在另一优选例中,所述的细胞包括:癌细胞或正常细胞; [0027] In another preferred embodiment, said cells include: cancer cells or normal cells;

[0028] 在另一优选例中,所述的细胞为肝癌细胞或肝细胞。 [0028] In another preferred embodiment, the cell is a liver cell or hepatocyte.

[0029] 在另一优选例中,所述方法还包括步骤: [0029] In another preferred embodiment, the method further comprising the steps of:

[0030] (b)对于步骤(a)中获得的候选化合物,进一步测试其对癌细胞生长或增殖的抑制作用。 [0030] (b) for a candidate compound of step (a) obtained, further testing its inhibitory effect on cell growth or proliferation.

[0031] 在另一优选例中,所述步骤(b)中包括步骤:测试组中,癌细胞的培养体系中添加测试化合物,并观察癌细胞的数量和/或生长情况;在对照组中,在癌细胞的培养体系中不添加测试化合物,并观察癌细胞的数量和/或生长情况;其中,如果测试组中癌细胞的数量或生长速度小于对照组,就表明该测试化合物是对癌细胞的生长或增殖有抑制作用的治疗癌症的候选化合物。 [0031] In another preferred embodiment, the step (b) includes the steps of: the test group, cancer cell culture system by adding the test compound, and observe the number and / or growth of cancer cells; in the control group In cancer cells in culture system without adding a test compound, and observe the number and / or growth of cancer cells; wherein, if the number of cancer cells in the test group or the growth rate than the control group, it indicates that the test compound is a cancer cell growth or inhibit the proliferation of cancer treatment candidate compound.

[0032] 本发明的第六方面,还提供了一种抑制或治疗癌症的方法,包括步骤:给需要治疗的对象(哺乳动物)施用安全有效量的SRRP35激动剂的用途。 [0032] The sixth aspect of the present invention, there is provided a method of inhibiting or treating cancer, comprising the steps of: to a subject in need of treatment (mammal) administering a safe and effective amount of SRRP35 agonist use.

[0033] 在另一优选例中,所述的癌症包括肝癌。 [0033] In another preferred embodiment, the cancer comprises liver.

[0034] 应理解,在本发明范围内中,本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合,从而构成新的或优选的技术方案。 [0034] It should be understood that within the scope of the present invention, can be combined with each other between the various technical features of the invention and the technical features below (as in Example) specifically described, so as to constitute a new or preferred technique program. 限于篇幅,在 Due to space limitations, in

此不再一一累述。 This not going tired. 附图说明 Brief Description

[0035] 图1A为实施例1中实时定量PCR检测SRRP35基因在32例肝癌病人癌组织和癌旁组织中的表达情况示意图,其中,“N”指癌旁组织,“C”指肝癌组织。 [0035] FIG. 1A in Example 1, quantitative real-time PCR to detect gene SRRP35 next 32 cases of hepatocellular carcinoma patients and cancer tissues map showing where, "N" refers to the adjacent tissues, "C" refers to the liver tissue. 结果显示20例(62.5%)病人的癌组织中SRRP35基因表达量低于相应的癌旁组织。 The results showed that 20 cases (62.5%) patients with carcinoma SRRP35 gene expression level lower than the corresponding adjacent tissues.

[0036] 图1B显示了实时定量PCR检测SRRP35基因在人肝癌细胞株中的表达模式。 [0036] Figure 1B shows real-time quantitative PCR detection SRRP35 gene in human hepatoma cell line expression patterns.

[0037] 图2A显示了SRRP35在肝癌细胞株Sk-1fep-1和WRL-68中的过表达情况,说明SRRP35在真核表达载体中成功表达。 [0037] FIG. 2A shows a hepatoma cell line Sk-1fep-1 and WRL-68 in the over-expression of SRRP35, described SRRP35 successfully expressed in eukaryotic expression vector.

[0038] 图2B显示了过表达的SRRP35蛋白减弱了肝癌细胞株Sk-Hep-1和WRL-68的克隆形成能力。 [0038] Figure 2B shows SRRP35 overexpressed protein weakens the colony formation hepatoma cell line Sk-Hep-1 and WRL-68's.

[0039] 图2C显示了过表达的SRRP35蛋白抑制Sk-1fep-1和WRL-68细胞的增值。 [0039] Figure 2C shows overexpressed protein inhibits SRRP35 value Sk-1fep-1 and WRL-68 cells.

[0040] 图3A显示了人工合成的SiRNA有效的干扰了SRRP35基因的表达。 [0040] FIG. 3A shows a synthetic SiRNA interferes with effective express SRRP35 genes.

[0041] 图3B显示了通过RNA干扰的方法沉默SRRP35的表达促进肝癌细胞Huh_7和H印3B的增值。 [0041] FIG. 3B shows a method of silencing by RNA interference expression promoting value-added SRRP35 hepatoma cell Huh_7 and H 3B of India.

[0042] 图4显示了通过RNA干扰的方法沉默SRRP35的表达提高了肝癌细胞Huh_7和Hep3B在软琼脂中的克隆形成能力,进而说明SRRP35抑制肝癌细胞的恶性表征。 [0042] Figure 4 shows the method of silencing by RNA interference expression SRRP35 improved Huh_7 and Hep3B hepatoma cell colony formation in soft agar, and then explain SRRP35 suppress malignant hepatoma cell characterization.

[0043] 图5A显示了过表达的SRRP35基因抑制肝癌细胞WRL-68在裸鼠皮下形成肿瘤的能力。 [0043] Figure 5A shows SRRP35 overexpression ability to inhibit liver cancer cells WRL-68 form tumors in nude mice. 无论在体积和肿瘤重量上SRRP35过表达组都比对照组小和轻,同时两组具有统计学意义。 Both in volume and tumor weight SRRP35 overexpression group than the control group, small and light, while the two groups was statistically significant.

[0044] 图5B显示了沉默SRRP35基因表达促进肝癌细胞Huh_7在裸鼠皮下形成肿瘤的能力。 [0044] FIG. 5B shows the ability to promote gene silencing SRRP35 Huh_7 liver cancer cells form tumors in nude mice. 无论在体积和肿瘤重量上SRRP35沉默表达组都比对照组大和重,同时两组具有统计学意义。 SRRP35 silence expression group than the control group in terms of large and heavy volume and tumor weight, while the two groups was statistically significant.

具体实施方式 DETAILED DESCRIPTION

[0045] 本发明人经过广泛而深入的研究,首次意外地发现,SRRP35在癌组织中低表达,而在癌旁组织及正常组织中高表达,因此SRRP35可作为癌症检测的标志物用于检测或辅助性检测癌症。 [0045] The present inventors have made extensive and intensive studies, for the first time unexpectedly found, SRRP35 low expression in cancer tissues and in adjacent tissues and high expression in normal tissues, thus SRRP35 can be used as a marker for cancer detection test or auxiliary detection of cancer. 此外,SRRP35的激动剂可抑制癌细胞(尤其是肝癌细胞)的生长。 In addition, SRRP35 agonists inhibit cancer cells (especially liver cells) growth. 在此基础上完成了本发明。 On this basis, the completion of the present invention.

[0046] 本发明人还以真核表达载体系统为介导系统,在肝癌细胞Sk-h印-1和WRL-68中过表达SRRP35基因(经鉴定过表达倍数为大于20倍)。 [0046] The present inventors have also eukaryotic expression vector system is mediated system in India hepatoma cells Sk-h-1 and WRL-68 SRRP35 overexpressed genes (identified overexpression multiple of more than 20 times). 通过细胞生长实验发现:SRRP35基因的过表达明显抑制肝癌细胞Sk-hep-Ι和WRL-68的生长。 Cell growth was found: SRRP35 gene overexpression significantly inhibited liver cancer cell Sk-hep-Ι and WRL-68 growth. 因此,可将SRRP35基因用于肝癌的基因治疗。 Thus, the gene may be SRRP35 gene therapy for liver cancer.

[0047] SRRP35蛋白和多核苷酸 [0047] SRRP35 protein and polynucleotides

[0048] 在本发明中,“本发明蛋白”、“本发明多肽”、“SRRP35蛋白”可互换使用,指简称为SRRP35)。 [0048] In the present invention, the "present invention protein", "polypeptide of the invention", "SRRP35 protein" are used interchangeably to refer to for short SRRP35). 应理解,所述术语还包括SRRP35的活性片段和衍生物。 It should be understood, the term also includes SRRP35 active fragments and derivatives thereof.

[0049] 在本发明中,“本发明基因”、“本发明多核苷酸”指编码SRRP35蛋白或其活性片段和衍生物的核苷酸序列,包括正义和反义核酸。 [0049] In the present invention, the "gene of the invention", "polynucleotide of the invention" refers to a nucleotide sequence encoding SRRP35 protein or active fragments and derivatives, including sense and antisense nucleic acid. SRRP35基因定位于细胞染色体6ql5,cDNA全长为786bp,编码全长261个氨基酸的蛋白。 SRRP35 gene is located on chromosome 6ql5, cDNA full length of 786bp, encoding a full length 261 amino acid protein.

[0050] 在本发明中,术语“SRRP35蛋白”、“SRRP35多肽”或“癌症标志物SRRP35”可互换使用,都指具有人蛋白SRRP35氨基酸序列的蛋白或多肽。 [0050] In the present invention, the term "SRRP35 protein", "SRRP35 polypeptide" or "Cancer Biomarkers SRRP35" are used interchangeably and all refer to a protein or polypeptide having the amino acid sequence of the protein human SRRP35. [0051] SRRP35,又名SRSF12 (serine/arginine-rich splicing factorl2,富含丝氨酸和精氨酸的剪切因子12),因最初发现作为SR的抑制因子且分子量为35kD而得名。 [0051] SRRP35, aka SRSF12 (serine / arginine-rich splicing factorl2, rich in serine and arginine shear factor 12), originally discovered as a result of SR inhibitor and a molecular weight of 35kD named. SR蛋白是一类富含丝氨酸和精氨酸的具有RRM RNA结合结构域的剪切因子,具有调控真核细胞pre-mRNA转录本选择性剪切掉内含子并拼接外显子的功能。 SR proteins are a class rich in serine and arginine binding domain of the shear factor has RRM RNA, can regulate eukaryotic pre-mRNA transcripts selectively cut out introns and exons spliced function. 体内研究发现,SRRP35能够拮抗其它SR蛋白并激活腺病毒ElA的pre-mRNA5'最末端的选择性剪切(Alison E, etal.2001.THE J0URNAL0F BIOLOGICAL CHEMISTRY)。 In vivo studies found that, SRRP35 can antagonize SR proteins and activate other adenoviral ElA of pre-mRNA5 'most end of the alternative splicing (Alison E, etal.2001.THE J0URNAL0F BIOLOGICAL CHEMISTRY).

[0052] SRRP35 基因的cDNA 序列如SEQ IDN0.:1 所示;Genbank 登录号135295,SRRP35 的cDNA序列CCDS47459.1以及其编码的氨基酸序列NP542781.3。 [0052] SRRP35 cDNA sequence of the gene, such as SEQ IDN0: 1 shown; Genbank Accession No. 135295, SRRP35 CCDS47459.1 cDNA sequence and its amino acid sequence encoded NP542781.3..

[0053] SRRP35基因所编码蛋白的氨基酸序列如SEQ ID N0.:2所示。 [0053] SRRP35 gene encoding the amino acid sequence of the protein such as SEQ ID N0:. 2 FIG.

[0054] 如本文所用,“分离的”是指物质从其原始环境中分离出来(如果是天然的物质,原始环境即是天然环境)。 [0054] As used herein, "isolated" refers to material isolated from its original environment out (if it is a natural material, i.e., the original environment is the natural environment). 如活体细胞内的天然状态下的多核苷酸和多肽是没有分离纯化的,但同样的多核苷酸或多肽如从天然状态中同存在的其他物质中分开,则为分离纯化的。 Polynucleotide and polypeptide natural state within living cells is under no separation and purification, but the same polynucleotide or polypeptide such as the presence of other substances in order to separate from the natural state, was isolated and purified. [0055] 如本文所用,“分离的SRRP35蛋白或多肽”是指SRRP35蛋白基本上不含天然与其相关的其它蛋白、脂类、糖类或其它物质。 [0055] As used herein, "isolated SRRP35 protein or polypeptide" refers SRRP35 protein is substantially free of its other native proteins, lipids, carbohydrates or other materials related. 本领域的技术人员能用标准的蛋白质纯化技术纯化SRRP35蛋白。 Those skilled in the art can use standard protein purification techniques SRRP35 protein. 基本上纯的多肽在非还原聚丙烯酰胺凝胶上能产生单一的主带。 Substantially pure polypeptide in a non-reducing polyacrylamide gel to produce a single main band. 在本发明中,SRRP35蛋白包括融合蛋白和非融合蛋白。 In the present invention, SRRP35 proteins include fusion proteins and non-fusion proteins.

[0056] 本发明的多肽可以是重组多肽、天然多肽、合成多肽,优选重组多肽。 [0056] The polypeptides of the present invention may be a recombinant polypeptide, a natural polypeptide, a synthetic polypeptide, preferably a recombinant polypeptide. 本发明的多肽还可包括或不包括起始的甲硫氨酸残基。 Polypeptides of the present invention may also include or not include an initial methionine residue.

[0057] 本发明的多核苷酸可以是DNA形式或RNA形式。 Polynucleotides [0057] The present invention may be in the form of DNA or RNA form. DNA形式包括cDNA、基因组DNA或人工合成的DNA。 DNA forms include cDNA, genomic DNA or synthetic DNA. DNA可以是单链的或是双链的。 DNA can be single-stranded or double-stranded. DNA可以是编码链或非编码链。 DNA may be the coding strand or non-coding strand.

[0058] 编码SRRP35的成熟多肽的多核苷酸包括:只编码成熟多肽的编码序列;成熟多肽的编码序列和各种附加编码序列;成熟多肽的编码序列(和任选的附加编码序列)以及非编码序列。 Polynucleotide mature polypeptide [0058] coding SRRP35 include: only the coding sequence encoding the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequence; the coding sequence for the mature polypeptide (and optionally additional coding sequence) and non- coding sequence. 术语“编码多肽的多核苷酸”可以是包括编码此多肽的多核苷酸,也可以是还包括附加编码和/或非编码序列的多核苷酸。 The term "polynucleotide encoding a polypeptide" may comprise a polynucleotide encoding such polypeptide, may also be polynucleotides also include additional coding and / or non-coding sequence.

[0059] 本发明还涉及上述多核苷酸的变异体,其编码与本发明有相同的氨基酸序列的多肽或多肽的片段、类似物和衍生物。 [0059] The present invention also relates to the polynucleotide variants, which encodes a polypeptide or fragment of the present invention has the same amino acid sequence of a polypeptide, analogs and derivatives thereof. 此多核苷酸的变异体可以是天然发生的等位变异体或非天然发生的变异体。 This polynucleotide variants can be variants allelic variants of naturally occurring or non-naturally occurring. 这些核苷酸变异体包括取代变异体、缺失变异体和插入变异体。 These nucleotide variants include substitution variants, deletion variants and insertion variants. 如本领域所知的,等位变异体是一个多核苷酸的替换形式,它可能是一个或多个核苷酸的取代、缺失或插入,但不会从实质上改变其编码多肽的功能。 As known in the art, an allelic variant is an alternate form of a polynucleotide, it may be substituted with one or more nucleotides, deletions or insertions, but does not materially change the function of their encoded polypeptides.

[0060] 本发明还涉及与上述的序列杂交的核酸片段,包括正义和反义的核酸片段。 [0060] The present invention also relates to the aforementioned sequence hybridizing nucleic acid fragments, including sense and antisense nucleic acid fragments. 如本文所用,“核酸片段”的长度至少含15个核苷酸,较好是至少30个核苷酸,更好是至少50个核苷酸,最好是至少100个核苷酸以上。 As used herein, "nucleic acid fragment" having a length of at least 15 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, preferably at least 100 nucleotides or more. 核酸片段可用于核酸的扩增技术(如PCR)以确定和/或分离编码SRRP35蛋白的多核苷酸。 Nucleic acid fragments can be used nucleic acid amplification techniques (e.g. PCR) to identify and / or isolate polynucleotides encoding SRRP35 protein.

[0061] 本发明的人SRRP35核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。 [0061] The present invention who SRRP35 full-length nucleotide sequences or fragments thereof are often used PCR amplification, recombinant method or synthetic method to obtain. 对于PCR扩增法,可根据已公开的有关核苷酸序列,尤其是开放阅读框序列来设计引物,并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板,扩增而得有关序列。 For PCR amplification, according to the relevant nucleotide sequences have been published, in particular open reading frame sequence to design primers and a commercially available cDNA library or cDNA library conventional methods known to those skilled in the as prepared by template, to amplify the relevant sequence derived. 当序列较长时,常常需要进行两次或多次PCR扩增,然后再将各次扩增出的片段按正确次序拼接在一起。 When using a long sequence, it is often the need for PCR amplification of two or more times, and then each time the amplified fragments spliced together in the correct order.

[0062] 一旦获得了有关的序列,就可以用重组法来大批量地获得有关序列。 [0062] Once the relevant sequence, you can use large quantities of recombinant methods to obtain information about the sequence. 这通常是将其克隆入载体,再转入细胞,然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。 This is typically cloned into a vector, then transferred to the cells, and then isolated from the host cell proliferation related sequence after by conventional methods.

[0063] 此外,还可用人工合成的方法来合成有关序列,尤其是片段长度较短时。 [0063] In addition, also with a synthetic approach to the synthesis sequence, in particular fragment length is short. 通常,通过先合成多个小片段,然后再进行连接可获得序列很长的片段。 Typically, the first synthesis of a number of small fragments, and then the long fragment obtained sequence.

[0064] 应用PCR技术扩增DNA/RNA的方法被优选用于获得本发明的基因。 [0064] Application of PCR amplified DNA / RNA is the preferred method for obtaining the gene of the present invention. 用于PCR的引物可根据本文所公开的本发明的序列信息适当地选择,并可用常规方法合成。 Primers for PCR can be appropriately selected depending on the sequence information disclosed herein present invention and synthesized by conventional methods. 可用常规方法如通过凝胶电泳分离和纯化扩增的DNA/RNA片段。 By conventional methods such as by gel electrophoresis and purified amplified DNA / RNA fragment.

[0065] 本发明也涉及包含本发明的多核苷酸的载体,以及用本发明的载体或SRRP35蛋白编码序列经基因工程产生的宿主细胞,以及经重组技术产生本发明所述多肽的方法。 [0065] The present invention also relates to compositions comprising a polynucleotide of the present invention carrier, as well as host cells or SRRP35 carrier protein coding sequence of the present invention is produced by genetic engineering, and a method of producing a polypeptide according to the invention by recombinant techniques.

[0066] 通过常规的重组DNA技术,可利用本发明的多核苷酸序列可用来表达或生产重组的SRRP35蛋白。 [0066] by conventional recombinant DNA techniques, a polynucleotide sequence can be used in the present invention may be used to express or produce recombinant proteins SRRP35. 一般来说有以下步骤: In general the following steps:

[0067] (I).用本发明的编码人SRRP35蛋白的多核苷酸(或变异体),或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞; . [0067] (I) of the present invention encoding human protein SRRP35 polynucleotide (or variant), or with a recombinant polynucleotide containing the expression vector transformed or transduced suitable host cell;

[0068] (2).在合适的培养基中培养的宿主细胞; . [0068] (2) cultured in a suitable medium, a host cell;

[0069] (3).从培养基或细胞中分离、纯化蛋白质。 [0069] (3) isolated from the medium or cells, the purified protein.

[0070] 本领域的技术人员熟知的方法能用于构建含人SRRP35编码DNA序列和合适的转录/翻译控制信号的表达载体。 [0070] Those skilled in the art known methods can be used to construct containing human DNA sequence coding SRRP35 and appropriate transcriptional / translational control expression vector signal. 这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。 These methods include in vitro recombinant DNA techniques, DNA synthesis, in vivo recombination technology. 所述的DNA序列可有效连接到表达载体中的适当启动子上,以指导mRNA合成。 The DNA sequence may be operably linked into an expression vector appropriate promoter to direct mRNA synthesis. 表达载体还包括翻译起始用的核糖体结合位点和转录终止子。 The expression vector also includes a translation initiation ribosome binding site and the use of transcription terminator.

[0071] 此外,表达载体优选地包含一个或多个选择性标记基因,以提供用于选择转化的宿主细胞的表型性状,如真核细胞培养用的二氢叶酸还原酶、新霉素抗性以及绿色荧光蛋白(GFP),或用于大肠杆菌的四环素或氨苄青霉素抗性。 [0071] In addition, the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as eukaryotic cell culture with dihydrofolate reductase, neomycin anti- resistance and green fluorescent protein (GFP), or for tetracycline or ampicillin resistance in E. coli.

[0072] 包含上述的适当DNA序列以及适当启动子或者控制序列的载体,可以用于转化适当的宿主细胞,以使其能够表达蛋白质。 [0072] The appropriate DNA sequence comprising said promoter and a suitable carrier or control sequence, may be used to transform a suitable host cell, to enable them to express the protein.

[0073] 宿主细胞可以是原核细胞,如细菌细胞;或是低等真核细胞,如酵母细胞;或是高等真核细胞,如哺乳动物细胞。 [0073] The host cell can be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. 代表性例子有:大肠杆菌,链霉菌属的细菌细胞;真菌细胞如酵母;植物细胞;果蝇S2或Sf9的昆虫细胞;CH0、COS、或293细胞的动物细胞等。 Representative examples include: E. coli, Streptomyces bacterial cells; fungal cells such as yeast; plant cell; Drosophila S2 or Sf9 insect cells; CH0, COS, 293 cells, or animal cells.

[0074] 用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。 [0074] a host cell transformed with the recombinant DNA can be used well known to those skilled in conventional techniques. 当宿主为原核生物如大肠杆菌时,能吸收DNA的感受态细胞可在指数生长期后收获,用CaCl2法处理,所用的步骤在本领域众所周知。 When the host is a prokaryote such as E. coli, can absorb the DNA of competent cells harvested after exponential growth phase with CaCl2 treatment, procedure used is well known in the art. 另一种方法是使用MgCl2。 Another method is to use MgCl2. 如果需要,转化也可用电穿孔的方法进行。 If desired, the conversion can be electroporation methods. 当宿主是真核生物,可选用如下的DNA转染方法:磷酸钙共沉淀法,常规机械方法如显微注射、电穿孔、脂质体包装等。 When the host is a eukaryote, it can be used as the DNA transfection methods: calcium phosphate precipitation method, conventional mechanical methods such as microinjection, electroporation, liposomes packaging.

[0075] 获得的转化子可以用常规方法培养,表达本发明的基因所编码的多肽。 [0075] The transformant obtained can be cultured by a conventional method, the gene of the present invention is the expression of the encoded polypeptide. 根据所用的宿主细胞,培养中所用的培养基可选自各种常规培养基。 Depending on the host cell used, the culture medium used may be selected from various conventional culture media. 在适于宿主细胞生长的条件下进行培养。 Cultured under conditions suitable for host cell growth. 当宿主细胞生长到适当的细胞密度后,用合适的方法(如温度转换或化学诱导)诱导选择的启动子,将细胞再培养一段时间。 When the host cells are grown to an appropriate cell density, the use of suitable methods (such as temperature conversion or chemical induction) induces the selected promoter, the cells were cultured for a period of time.

[0076] 在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。 [0076] In the above method, the recombinant polypeptide may be in the cells, or the expression on the cell membrane or secreted outside the cells. 如果需要,可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。 If necessary, use its physical, chemical, and other properties of the separation and purification of recombinant proteins through various separation methods. 这些方法是本领域技术人员所熟知的。 These methods are to those skilled in the art. 这些方法的例子包括但并不限于:常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结 Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment (salting out method) protein precipitant, centrifugation, osmotic breaking strain, super treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer precipitation, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatographic techniques and methods of those nodes

口ο Mouth ο

[0077] 抗体 [0077] Antibody

[0078] 本发明还包括对人SRRP35蛋白具有特异性的多克隆抗体和单克隆抗体,尤其是单克隆抗体。 [0078] The present invention also includes human SRRP35 protein specific polyclonal and monoclonal antibodies, especially monoclonal antibodies. 这里,“特异性”是指抗体能结合于人SRRP35基因产物或片段。 Here, "specific" refers to an antibody capable of binding to human SRRP35 gene product or fragments thereof. 较佳地,指那些能与人SRRP35基因产物或片段结合但不识别和结合于其它非相关抗原分子的抗体。 Preferably, those with human SRRP35 gene product or fragment binds to but does not recognize and bind to other non-associated antigen antibody molecules. 本发明的抗体可以通过本领域内技术人员已知的各种技术进行制备。 Antibodies of the invention can be prepared by techniques known in the art various techniques.

[0079] 本发明不仅包括完整的单克隆或多克隆抗体,而且还包括具有免疫活性的抗体片段,如Fab'或(Fab)2片段;抗体重链;抗体轻链;遗传工程改造的单链Fv分子;或嵌合抗体。 [0079] The present invention encompasses not only intact monoclonal or polyclonal antibodies, but also immunologically active antibody fragments, such as Fab 'or (Fab) 2 fragment; antibody heavy chain; antibody light chain; genetically engineered single chain Fv molecule; or a chimeric antibody.

[0080] 抗人SRRP35蛋白的抗体可用于免疫组织化学技术中,检测活检标本中的人SRRP35 蛋白。 [0080] anti-human SRRP35 protein antibodies can be used in immunohistochemical techniques to detect human biopsy specimens SRRP35 protein.

[0081] 激动剂和药物组合物 [0081] agonists and pharmaceutical compositions

[0082] 利用本发明蛋白,通过各种常规筛选方法,可筛选出与SRRP35蛋白发生相互作用的物质,尤其是激动剂等。 [0082] The use of the present invention protein by various conventional screening methods can be selected with SRRP35 protein interaction occurring substances, especially agonists like.

[0083] 本发明SRRP35蛋白的激动剂,当在治疗上进行施用(给药)时,可促进SRRP35蛋白的表达和/或活性,进而抑制癌细胞(包括肝癌)的生长或增殖。 [0083] The present invention SRRP35 protein agonists, when administered (administration) in the treatment, it can promote the expression and / or activity of SRRP35 protein, thereby inhibiting cancer cells (including liver cancer) growth or proliferation. 通常,可将这些激动剂配制于无毒的、惰性的和药学上可接受的水性载体介质中,其中PH通常约为5-8,较佳地pH约为6-8,尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。 Typically, such agonists can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein PH is usually about 5-8, preferably about pH 6-8, although pH value may vary formulated nature of the substance and the condition to be treated varies. 配制好的药物组合物可以通过常规途径进行给药,其中包括(但并不限于):瘤内、肌内、腹膜内、静脉内、皮下、皮内、或局部给药。 Formulated pharmaceutical compositions may be administered by conventional routes, including (but not limited to): intratumoral, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal or topical administration.

[0084] 本发明还提供了一种药物组合物,它含有安全有效量的本发明SRRP35蛋白或其激动剂以及药学上可接受的载体或赋形剂。 [0084] The present invention also provides a pharmaceutical composition comprising a safe and effective amount of a protein or SRRP35 agonist and a pharmaceutically acceptable carrier or excipient of the present invention. 这类载体包括(但并不限于):盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。 Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. 药物制剂应与给药方式相匹配。 The pharmaceutical formulation phase should match the mode of administration. 本发明的药物组合物可以被制成针剂形式,例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。 The pharmaceutical compositions of the present invention may be prepared in the form of injections, such as with saline or an aqueous solution containing glucose and other adjuvants were prepared by conventional methods. 诸如片剂和胶囊之类的药物组合物,可通过常规方法进行制备。 The pharmaceutical compositions such as tablets and capsules and the like, can be prepared by conventional methods. 药物组合物如针剂、溶液、片剂和胶囊宜在无菌条件下制造。 Pharmaceutical compositions such as injections, solutions, tablets and capsules should be manufactured under sterile conditions. 活性成分的给药量是治疗有效量,例如每天约I微克-10毫克/千克体重。 The amount of active ingredient administered is a therapeutically effective amount, e.g., about I microgram per day -10 mg / kg body weight.

[0085] 检测方法和试剂盒 [0085] detection method and kit

[0086] 本发明还涉及定量和定位检测人SRRP35蛋白水平或mRNA水平的诊断试验方法。 [0086] The present invention also relates to a method of locating quantitative and diagnostic test to detect human SRRP35 protein level or mRNA level. 这些试验是本领域所熟知的。 These tests are known in the art. 试验中所检测的人SRRP35蛋白水平,可以用于诊断肝癌。 SRRP35 protein levels detected in human trials, may be used to diagnose liver cancer.

[0087] 一种检测样品中是否存在SRRP35蛋白的方法是利用SRRP35蛋白的特异性抗体进行检测,它包括:将样品与SRRP35蛋白特异性抗体接触;观察是否形成抗体复合物,形成了抗体复合物就表示样品中存在SRRP35蛋白。 [0087] protein in the presence or absence of a test sample SRRP35 method is the use of specific antibodies SRRP35 protein detection, comprising: contacting the sample with antibodies specific contacting SRRP35 protein; antibody complex formation is observed, the antibody complex is formed says SRRP35 protein present in the sample.

[0088]SRRP35蛋白或其多核苷酸可用于SRRP35蛋白相关疾病的诊断和治疗。 [0088] SRRP35 protein or polynucleotide may be used to diagnose and treat SRRP35 protein-related diseases. 本发明的多核苷酸的一部分或全部可作为探针固定在微阵列或DNA芯片上,用于分析组织中基因的差异表达分析和基因诊断。 Polynucleotide or all part of the invention can be used as probes immobilized on a microarray or DNA chip analysis for differential gene expression analysis of tissues and genetic diagnosis. 抗SRRP35的抗体可以固定在蛋白质芯片上,用于检测样品中的SRRP35 蛋白。 Anti SRRP35 antibody may be immobilized on the protein chip, for detecting the sample SRRP35 protein.

[0089] 本发明还提供了一种检测肝癌的试剂盒,它含有特异性扩增SRRP35的引物对和/或SRRP35特异性抗体。 [0089] The present invention also provides a kit for the detection of liver cancer, which contains specific amplification SRRP35 primer pairs and / or SRRP35 specific antibodies.

[0090] 筛选方法 [0090] Screening Method

[0091] 本发明还提供了基于SRRP35进行药物筛选的方法。 [0091] The present invention also provides a method for drug screening based SRRP35. 一种方法是先筛选影响(促进)SRRP35表达或活性的化合物,然后对筛选出的化合物进一步测试其对癌细胞。 One method is to filter effect (promote) SRRP35 expression or activity of the compounds, and then screening compounds further tested on cancer cells. 一种筛选方法可基于SRRP35的mRNA的表达水平。 A screening method may be based on expression levels of mRNA of SRRP35.

[0092] 其中,代表性的癌细胞包括(但并不限于):肝癌细胞。 [0092] wherein, cancer cells typically include (but are not limited to): hepatoma cells.

[0093] 通用方法: [0093] General Methods:

[0094] (I)临床组织样本的获取 [0094] (I) clinical tissue samples to obtain

[0095] 肝癌及癌旁组织取自手术治疗的肝癌患者,在获取样本前均与患者签订了知情同意书。 [0095] next to the liver and tumor tissue from surgical treatment of liver cancer patients, with patients before obtaining samples are signed informed consent. 手术切除的肝脏一经离体,迅速切取肿瘤原发灶及周围5cm以外的癌旁组织,投入液氮中速冻并移至一80C冰箱保存,运输时储存于液氮中。 Surgical resection of liver upon in vitro, rapid cut in and around the original tumor foci than 5cm adjacent tissue, frozen in liquid nitrogen and transferred to a 80 C refrigerator, stored in liquid nitrogen during transport. 癌与癌旁组织均通过病理专家做出最终诊断。 Cancer and adjacent tissues are to make the final diagnosis by pathologists. 样本依据Edmondson分级标准分为1-1I I级。 Edmondson grading standards based sample is divided into 1-1I I.

[0096] (2)组织及细胞RNA抽提 [0096] (2) tissue and cell RNA extraction

[0097] 采用TRIzol Reagent (Invitrogen)试剂抽提RNA,具体操作如下: [0097] using TRIzol Reagent (Invitrogen) reagent extracted RNA, do the following:

[0098] I)研钵、碾杵和匀浆器等器皿洗净,分别再用ddH20和DEPC H2O冲洗,然后在180C烘箱中烘约4小时,以去除RNA酶; Wash utensils [0098] I) mortar and pestle and grind homogenizer, etc., were then ddH20 and DEPC H2O wash, then bake for about four hours at 180 C in an oven to remove the RNA enzyme;

[0099] 2)在研钵中加入适量液氮使之预冷,将组织从液氮中迅速取出,切取约50-1OOmg大小,在研钵中研磨成粉末; [0099] 2) by adding an appropriate amount of liquid nitrogen in a mortar to make the pre-cooling, the organization quickly removed from the liquid nitrogen, cut approximately 50-1OOmg size, grind in a mortar to a powder;

[0100] 3)用刮匙将研磨好的组织粉末尽可能完全的移至无RNA酶的EP管中,EP管被预先加入适量体积(Iml) TRIzoI试剂,充分匀浆; [0100] 3) with a curette good organization milled powder as completely as possible to move without RNA enzyme EP tube, EP tube is added to the appropriate volume beforehand (Iml) TRIzoI reagent fully homogenized;

[0101] 4)室温放置5分钟,按比例向离心管中加入氯仿(200μ 1/lml TRIzol),迅速剧烈振荡15秒,室温静置2-3分钟,4C, 12000Xg条件下尚心15分钟; [0101] 4) at room temperature for 5 minutes, according to the proportion of chloroform was added to the centrifuge tube (200μ 1 / lml TRIzol), quickly shake vigorously for 15 seconds, at room temperature for 2-3 min, 4 C, still under 12000Xg heart condition 15 minutes;

[0102] 5)将上层水相尽可能地转移至新的无RNA酶的EP管中,加入等体积的异丙醇,颠倒混匀5次,室温静置10分钟,4C,12000 X g条件下离心10分钟,此时可见RNA沉淀; [0102] 5) the upper aqueous phase was transferred to a possible new non-RNA enzyme EP tube, add an equal volume of isopropanol, mix by inversion 5 times, at room temperature for 10 minutes, 4 C, 12000 X g under the conditions centrifuged for 10 minutes at which time RNA pellet was visible;

[0103] 6)将上清倒掉,加入75%乙醇(lml/lml TRIzol),混匀,洗涤RNA,离心4C,7500 X g条件下离心5分钟; [0103] 6) The supernatant was drained, add 75% ethanol (lml / lml TRIzol), mixed and centrifuged for 5 minutes at washing RNA, centrifuged at 4 C, 7500 X g conditions;

[0104] 7)弃上清,尽可能除尽残留乙醇,沉淀自然干燥5-10min (注意切勿完全干燥);加入30 - 50 μ I DEPC H2O,吹吸几次,溶解RNA沉淀; [0104] 7) The supernatant was discarded, as divisible residual ethanol precipitate natural drying 5-10min (Be careful not to completely dry); adding 30 - 50 μ I DEPC H2O, pipetting several times to dissolve RNA precipitation;

[0105] 8)酶标仪测定RNA浓度及纯度0D260/280( 1.8 - 2.0);凝胶电泳观察有无降解,一80C保存。 [0105] 8) then determined RNA concentration and purity of 0D260 / 280 (1.8 - 2.0); gel electrophoresis to observe whether the degradation, a 80 C to save.

[0106] 细胞株RNA抽提,取对数生长期的细胞,吸取培养液,根据培养皿的面积加入相应量的TRIzol试剂(lml TRIzol/lOcm2)裂解细胞,吹打几次,将裂解下来的细胞收集至无RNA酶的EP管中,其余按照上述步骤4) -8)完成氯仿-异丙醇法分离纯化RNA。 [0106] cell lines RNA extraction logarithmic growth phase cells absorb broth, add the appropriate amount of TRIzol reagent (lml TRIzol / lOcm2) according to dish area cell lysis by pipetting several times, the cleaved cell No RNA enzyme was collected to EP tube, and the rest according to the above step 4) -8) completion of chloroform - isopropanol were separated and purified RNA.

[0107] (3) RNA的逆转录 [0107] (3) RNA reverse transcription

[0108]用 M-MLV Reverse Transcriptase (Promega)逆转录,操作如下: [0108] The M-MLV Reverse Transcriptase (Promega) reverse transcriptase, as follows:

[0109] I)在无核酸酶的EP管中加入以下组分: [0109] I) added nuclease-free EP tube the following components:

[0110] [0110]

Figure CN103937871AD00101

[0111] 置于PCR仪中,70C,5分钟,然后立即在冰上冷却5min。 [0111] placed in PCR instrument, 70 C, 5 minutes and then immediately cooled on ice 5min.

[0112] 2)在上述体系中再加入以下组分: [0112] 2) In the above system, then add the following components:

[0113] [0113]

Figure CN103937871AD00102

[0114] 轻轻混匀后,置于PCR仪中,370C,60min。 After the [0114] Mix gently placed in PCR instrument, 370C, 60min.

[0115] 逆转得到的cDNA置于4C保存。 [0115] The resulting cDNA reversed placed at 4 C.

[0116] (4)实时定量PCR [0116] (4) Real-time quantitative PCR

[0117]实时定量 PCR 反应使用SYBR* Premix ExTaq™ (Perfect RealTime)试剂盒(TaKaRaBiotechnology C0., Ltd.Dalian, China)的反应体系,利用Thermal CyclerDice™RealTime System (TP800实时荧光定量PCR仪,TaKaRa)进行操作。 [0117] Real-time quantitative PCR reactions using SYBR * Premix ExTaq ™ (Perfect RealTime) kit (TaKaRaBiotechnology C0., Ltd.Dalian, China) reaction system using Thermal CyclerDice ™ RealTime System (TP800 real-time PCR instrument, TaKaRa) operation. 定量PCR的扩增产物长度以80bp - 150bp最为合适(可以延长至300bp)。 Quantitative PCR amplified product length to 80bp - 150bp most appropriate (can be extended to 300bp).

[0118] 反应体系如下: [01] The reaction system is as follows:

[0119] [0119]

Figure CN103937871AD00103

[0121] 溶解曲线分析步骤: [0121] melting curve analysis step:

[0122] 95 C 15sec [0122] 95 C 15sec

[0123] 60 C 30sec [0123] 60 C 30sec

[0124] 95 C 15sec[0125] 解离时间为4sec。 [0124] 95 C 15sec [0125] dissociation time was 4sec.

[0126] 荧光本底信号和阈值采用仪器设置的默认值,每次PCR反应结束后会自动生成,Ct值表示每个反应管内的荧光信号达到设定阈值(基线荧光强度的10倍)时所经历的循环数;目的基因SRRP35每个模板做3个复管,得到的Ct值取平均值;SRRP35基因的Ct平均值减去相应模板的内参基因(β-actin)的Ct平均值,得到ACt。 When the [0126] fluorescence background signal and the threshold value with the default value instrument settings are automatically generated after each PCR reaction, Ct value represents the fluorescence signal of each reaction tube reaches the set threshold (10 times the baseline fluorescence intensity) of the the number of cycles experienced; target gene SRRP35 each template to do three complex tube, Ct values obtained by averaging; Ct mean SRRP35 reference gene is subtracted gene corresponding template (β-actin) of Ct mean, get ACt . 肝癌组的ACt减去相应癌旁组织的ACt,得到Λ Λ Ct值,肝癌组和癌旁组中的SRRP35基因的倍数关系用2_“^表 ACt minus HCC corresponding adjacent tissues ACt, get Λ Λ Ct value, multiple relationships and cancer adjacent liver cancer group SRRP35 gene was 2 _ "^ table

/Jn ο / Jn ο

[0127] (5)真核表达载体构建 [0127] (5) eukaryotic expression vector

[0128] I)模板:人肝永生化细胞L02的cDNA文库。 [0128] I) template: human liver L02 immortalized cell cDNA library.

[0129] 2)真核表达载体的选择:pcDNA™3.1/myc-His (-) A, 5522nucleotides。 [0129] 2) eukaryotic expression vector of choice: pcDNA ™ 3.1 / myc-His (-) A, 5522nucleotides.

[0130] 3)根据SRRP35mRNA (NM080743.4)序列,结合表达载体pcDNA™3.1/myc-His (-) A的酶切位点设计引物,引物序列如SEQ ID N0.:3 (正向)以及SEQIDN0.:4 (反向)所示。 . [0130] 3) The SRRP35mRNA (NM080743.4) sequence, combined with the expression vector pcDNA ™ 3.1 / myc-His (-) A restriction sites designed primers, primer sequences as SEQ ID N0: 3 (forward) and SEQIDN0:. 4 (reverse) of Fig. 其中反向引物中SRRP35的终止密码子被去除,使得SRRP35的C末端带上c-myc和6xHi s标签。 Wherein the reverse primer SRRP35 stop codon is removed, so that the C-terminal SRRP35 belt of c-myc and 6xHi s label. 利用高保真性DNA 聚合酶PrimeSTAR™HS DNA Polymerase (TaKaRa),以L02cDNA 为模板扩增基因SRRP35全长开放读码框,50 μ I总反应体系成分如下: The use of high-fidelity DNA polymerase PrimeSTAR ™ HS DNA Polymerase (TaKaRa), to L02cDNA as template to amplify the full length gene SRRP35 open reading frame, 50 μ I of the total reaction system components as follows:

[0131] [0131]

Figure CN103937871AD00111

[0132] 采用两步PCR法(98 C,IOsec ;60C,90sec),扩增35个循环。 [0132] The two-step PCR method (98 C, IOsec; 60 C, 90sec), 35 cycles of amplification. PCR产物大小约 About the size of the PCR product

0.8kb,1%琼脂糖凝胶电泳鉴定大小,割胶回收(胶纯化试剂盒:MACHEREY-NAGEL)符合片段大小的PCR产物。 0.8kb, 1% agarose gel electrophoresis to identify the size, tapping recovered (gel purification kit: MACHEREY-NAGEL) in line with the size of the PCR fragments.

[0133] EcoRV, Hind III (TaKaRaBiotechnology Inc.Dalian, China)双酶切回收PCR 产物及载体质粒pcDNA™3.1/myc-His (-) A,酶切反应体系如下: [0133] EcoRV, Hind III (TaKaRaBiotechnology Inc.Dalian, China) Recycling digested PCR products and plasmid pcDNA ™ 3.1 / myc-His (-) A, digestion reaction as follows:

Figure CN103937871AD00112

[0135] 37C酶切反应I小时;割胶回收酶切产物。 [0135] 37 C digestion reaction I hour; tapping recovered digestion products. [0136] 4)连接:酶切回收的PCR产物与载体按照摩尔数比(4:1)的比例混合,DNA连接酶体系链接,体系中还包括2.5μ 14XSolution I (TaKaRa Code:D102A), ddH20补齐至10μ 1,16C连接2h乃至过夜; [0136] 4) is connected: The PCR product was digested with the recovered carrier in accordance with the number of molar ratio (4: 1 mix) ratio, DNA ligase link system, the system further comprising 2.5μ 14XSolution I (TaKaRa Code: D102A), ddH20 filled to 10μ 1,16 C overnight and the connection 2h;

[0137] 5)转化:取10 μ I连接产物与100 μ I感受态细菌(Τ0Ρ10或DH5 α )混合,冰上放置30min,42C热激90sec,立即置于冰上5min,加入800 μ I不含抗生素的LB培养液,37C、200rpm振荡培养30min,使菌体复苏且扩增一代,3000rpm离心2min,去除大部分上清,留50 - 100 μ I菌液,,轻轻吹打沉淀混匀,然后均匀涂于有氨苄青霉素抗性(Amp+)的LB平板上,37C培养12 - 16小时。 [0137] 5) Conversion: Take 10 μ I connect product with 100 μ I competent bacteria (Τ0Ρ10 or DH5 α) mixed, placed on ice for 30min, 42 C heat shock 90sec, immediately placed on ice for 5min, added 800 μ I antibiotic-free LB broth, 37 C, 200rpm shaking culture 30min, so that the cell recovery and amplification generation, 3000rpm centrifuge 2min, remove most of the supernatant, leaving 50 - 100 μ I gently pipetting bacteria ,, precipitation mix, then evenly applied to have the ampicillin resistance (Amp +) LB plate, 37 C culture 12--16 hours.

[0138] 6)克隆鉴定:挑取经过氨苄抗性筛选后生长的菌落在加氨苄青霉素的液体培养基中扩大培养,抽取质粒进行酶切鉴定:取1- 2 μ g小抽质粒用EcoRV,Hind III双酶切,琼脂糖凝胶电泳鉴定酶切片段大小,载体pcDNA™3.1/myc-His (_) A片段大小约5.5kb,SRRP35读码框片段大小约800bp,符合大小的克隆送测序确认插入片段序列的正确性。 [0138] 6) clone identification: After picked ampicillin-resistant colonies screened expand growth in Canada ampicillin liquid culture medium, a plasmid identified by restriction enzyme extract: Take 1- 2 μ g small pumping plasmids EcoRV, Hind III restriction enzyme digestion, agarose gel electrophoresis fragment size, vector pcDNA ™ 3.1 / myc-His (_) A fragment size of about 5.5kb, SRRP35 ORF fragment size of about 800bp, in line with the size of the clones sequenced delivery confirm the correctness insert fragment sequences.

[0139] (6)细胞生长曲线的测定 [0139] (6) Determination of cell growth curve

[0140] I)将不同种类的HCC细胞根据其生长特性按3-5X 103/100 μ I/孔计算细胞总量,充分消化细胞后,稀释到所需浓度,接种于96孔板内。 After the [0140] I) the different types of cells to calculate the total amount of HCC cells according to their growth characteristics by 3-5X 103/100 μ I / hole, to fully digest the cells, diluted to the desired concentration, were seeded in 96-well plates. 每天每组三复孔,按5 - 7天接种细胞; Groups of three wells per day, according to 5--7 days after inoculation cell;

[0141] 2)待细胞基本贴壁后观察细胞状态和数目。 [0141] 2) pending state and the number of cells adherent after basic observation. 用CCK-8显色剂(CellCountingKit-8, D0JIND0, Japan)进行显色反应,每100 μ I 培养液加10 μ I CCK-8,37C,5%C02培养箱放置孵育lh,酶标仪测定450nm处的吸光度,记录,确定细胞实际的起始密度,作为生长相对零点。 Color reaction with CCK-8 reagent (CellCountingKit-8, D0JIND0, Japan), per 100 μ I medium plus 10 μ I CCK-8,37 C, 5% C02 incubator placed incubated lh, ELISA The absorbance was measured at 450nm, recording, determine the actual starting cell density, as relative zero growth.

[0142] 3)每天或隔天半量换液,具体视实验要求而定; [0142] 3) or the next day and a half per day was changed, depending on the test requirements may be;

[0143] 4)显微镜下观察细胞形态,固定时间间隔测量,记录细胞生长状况; Observation [0143] 4) microscope cell morphology, fixed time interval measurement, recording cell growth conditions;

[0144] 5) 一般测5至7天。 [0144] 5) General measure 5-7 days. 待测定结束后,收集数据进行处理,用Excel绘出图表。 Be measured after the collection of data processing, plotted with Excel chart.

[0145] (7)细胞克隆形成实验 [0145] (7) colony formation assay

[0146] I)转染:采用Lipofectamine™2000 (Invitrogen)转染细胞,过表达或沉默细胞中SRRP35基因的表达; [0146] I) Transfection: The Lipofectamine ™ 2000 (Invitrogen) transfected cells, over-expression or silencing gene expression in cells SRRP35;

[0147] 2)转染后的细胞,于6孔板或35mm培养皿用正常培养液培养24h后消化计数,按一定数目接种至IOOmm培养皿(不同细胞株数目不同),继续培养24h,然后依据细胞种类加入适当浓度的G418 (600 - 1000μ g/ml),以筛选转染阳性的细胞克隆; [0147] 2) transfected cells in 6-well plates or 35mm Petri dishes with normal culture medium 24h after digestion count, according to a certain number of inoculated IOOmm dishes (a different number of different cell lines), cultured 24h, then according to cell types by adding the appropriate concentration of G418 (600 - 1000μ g / ml), to screen transfected cells positive clones;

[0148] 3)培养2-3周,其间每隔3-5天更换新鲜培养液并加G418筛选,直至有肉眼可见的细胞克隆形成; [0148] 3) for 2-3 weeks, during which replaced with fresh medium and add G418 screening every 3-5 days until a visible cell colony formation;

[0149] 4)吸去培养皿内的培液,IXPBS洗两次,考马斯亮蓝R-250染色2h,用水轻轻冲洗后,再用考马斯亮蓝染色脱色液脱色30 - 60min ; [0149] 4) to absorb the culture liquid in a vessel, IXPBS washed twice with Coomassie Brilliant Blue R-250 staining 2h, wash gently with water and then stained with Coomassie brilliant blue destaining solution bleaching 30 - 60min;

[0150] 5)克隆形成染色结果拍照,按照相同的标准(细胞克隆大小)对每个培养皿上的细胞克隆进行计数。 [0150] 5) cloning staining pictures, according to the same criteria (cell clone size) was cloned cells were counted on each plate.

[0151] (8)蛋白质印迹(Western Blot) [0151] (8) Western blots (Western Blot)

[0152] I)蛋白样品制备:培养的细胞吸去培养上清后,用预冷的IXPBS洗两次,加入2X SDS裂解液(IOOmM Tri s_Cl,pH = 6.8,4%SDS,20%甘油),充分裂解后,沸水浴加热IOmin, 12000 Xg 离心IOmin,上清转移至新管中,Pierce' BCA ProteinAssayKit 对获得的蛋白进行定量,一80C保存; [0152] I) Preparation of protein samples: After withdrawing the cultured cells to the culture supernatant, washed twice with precooled IXPBS adding 2X SDS lysis buffer (IOOmM Tri s_Cl, pH = 6.8,4% SDS, 20% glycerol) After fully cracking, boiling water bath heating IOmin, 12000 Xg centrifugation IOmin, the supernatant was transferred to a new tube, Pierce 'BCA ProteinAssayKit protein obtained quantitatively, a 80 C preservation;

[0153] 2)蛋白电泳分离:在蛋白样品加入适量含有200mM DTT的上样缓冲液(loadingbuffer),沸水浴加热IOmin,稍作离心,SDS-PAGE蛋白凝胶电泳分离样品; [0153] 2) protein electrophoresis separation: the protein samples containing 200mM DTT adding an appropriate amount of loading buffer (loadingbuffer), boiling water bath for heating IOmin, a brief centrifugation, SDS-PAGE protein gel electrophoresis sample;

[0154] 3)转膜:将电泳胶、硝酸纤维膜、厚(薄)滤纸垫板浸于转膜缓冲液中(24mMTris,192mM甘氨酸,20%甲醇)平衡15_20min。 [0154] 3) transfer membrane: the electrophoresis gel, nitrocellulose membrane, thickness (thin) filter pad was immersed in transfer buffer (24mMTris, 192mM glycine, 20% methanol) equilibrated 15_20min. 按正极_ I层厚滤纸垫板_硝酸纤维膜_电泳胶-2层薄滤纸垫板-负极的顺序放好,湿转仪(XCellSureLock™, invitrogen) 30伏转膜30_40min ; According to the thickness of the filter plate positive _ I _ _ nitrocellulose membrane filter paper electrophoresis gel -2 thin plate - Negative sequence put away wet gyroscope (XCellSureLock ™, invitrogen) 30 volt transmembrane 30_40min;

[0155] 4)封闭:5%脱脂奶粉/0.P/oPBST作为封闭液,水平摇床,室温封闭30min_2h ; [0155] 4) Blocking: 5% skim milk as the blocking solution /0.P/oPBST horizontal shaker at room temperature closed 30min_2h;

[0156] 5) 一抗:一抗用封闭液稀释(参考抗体说明书推荐浓度),室温孵育2h或者4C孵育过夜,0.1%PBST洗三次,每次5min ; [0156] 5) antibody: antibody diluted in blocking solution (recommended specification reference antibody concentration), incubated 2h at room temperature or incubate overnight at 4 C, 0.1% PBST washed three times, each time 5min;

[0157] 6) 二抗:荧光二抗用封闭液稀释(1:1000),室温孵育301^11,0.1%PBST洗三次,每次5min ; [0157] 6) secondary antibody: fluorescent secondary antibody was diluted (in blocking buffer 1: 1000) was incubated at room temperature 301 ^ 11,0.1% PBST washed three times, each time 5min;

[0158] 7)扫膜:0DYSSEY红外成像系统扫描硝酸纤维素膜,保存图像。 [0158] 7) sweep membrane: 0DYSSEY infrared imaging system to scan a nitrocellulose membrane, save the image.

[0159] (9)软琼脂克隆形成实验 [0159] (9) soft agar colony formation assay

[0160] 1)分别配制1%和2%的低熔点Agarose (TaKaRa公司),高温高压灭菌; [0160] 1) were prepared 1% and 2% of the low melting point Agarose (TaKaRa Co.), autoclavable;

[0161 ] 2)配制2 X DMEM 培养液(2.5 X DMEM,含20%FBS); [0161] 2) Preparation of 2 X DMEM culture medium (2.5 X DMEM, containing 20% FBS);

[0162] 3)将37C保温的2%Agarose和2XDMEM培养液按相同体积混合,以每孔0.5ml加到24孔板中,置于4C冰箱中,待凝固后使用; [0162] 3) the 37 C heat the 2% Agarose and 2XDMEM the same volume of broth mixed with 0.5ml per well was added to 24-well plates, placed 4 C refrigerator to be solidified after use;

[0163] 4)充分消化培养的细胞成单个细胞,计数,稀释成相同浓度(3000-5000个/0.5ml); [0163] 4) fully digest cultured cells into a single cell, count, diluted to the same concentration (3000-5000 /0.5ml);

[0164] 5)将细胞悬液与37C保温的l%Agar0Se以相同体积混合,加到已经铺好下层胶的24孔板中,每孔0.5ml,置于4C冰箱IOmin ; [0164] 5) The cell suspension was incubated with 37 C in l% Agar0Se mixed with the same volume, has paved the lower gum was added to 24-well plates, each well 0.5ml, placed in 4 C refrigerator IOmin;

[0165] 6)在凝固的软琼脂上加入0.2ml培养液,置于37〇,5%0)2培养箱中,继续培养2-3周; [0165] 6) was added to the solidified soft agar 0.2ml broth, placed in 37 billion, 5% 0) 2 incubator, and cultured for 2-3 weeks;

[0166] 7)显微镜下观察每个孔里面细胞克隆的生长情况,计数,进行分析。 Growth was observed inside the cell clone in each well at [0166] 7) microscope, counting, were analyzed.

[0167] (10)裸鼠成瘤实验 [0167] (10) in Nude Mice

[0168] I)所用小鼠为5-6周大小的雄性BLAB/c nu裸鼠,由上海斯莱克实验动物有限公司提供,饲养于南方模式动物培养中心; [0168] I) used mice 5-6 weeks old male BLAB / c nu mice, Ltd. on Hayes Lake by animals, feeding an animal model development center in the south;

[0169] 2)取处理后的细胞,以相同数量接种于小鼠皮下(不同细胞种类接种数目不同),为避免个体差异导致的误差,同种细胞不同处理可左右对称接种于同一只小鼠; [0169] 2) treatment of the cells to take in the same amount was inoculated subcutaneously to mice (a different number of different cell types seeded), in order to avoid errors due to individual differences, the same kinds of cells can be treated in the same symmetrical inoculated mice ;

[0170] 3)待出现可见肿瘤后,每3天监测肿瘤大小,游标卡尺读取肿瘤长径和短径, [0170] 3) appear to be visible tumor after every three days to monitor tumor size, tumor caliper read long and short diameters,

[0171] 按以下公式计算肿瘤体积:体积=长径X短径2 ; [0171] Tumor volume was calculated according to the following formula: Volume = length of the minor axis diameter X 2;

[0172] 4)持续监测约7-8次后,整理数据,统计。 [0172] 4) continuous monitoring of about 7-8 times, organize data and statistics.

[0173] (11)抗体获得和免疫检测 [0173] (11) antibodies and immunoassays to obtain

[0174] I)抗原蛋白获得 [0174] I) antigen protein obtained

[0175] 从Genebank数据库中获得人SRRP35基因的cDNA序列,通过PCR扩增获得编码框,插入原核生物或真核生物表达载体中,表达SRRP35蛋白,并按基因工程表达产物的纯化体系纯化蛋白。 [0175] obtained from the Genebank database SRRP35 cDNA sequence of the human gene, obtained by PCR amplification coded frame, insert prokaryotic or eukaryotic expression vector, expression SRRP35 protein expression products of genetic engineering in accordance with the purified protein purification system.

[0176] 2)抗体制备[0177] 可采用以下几种方法制备抗体: [0176] 2) Antibody [0177] Antibodies can be prepared in several ways:

[0178] a细胞融合法:用上述制备的SRRP35蛋白免疫动物(包括兔子、山羊等),获得脾脏细胞,再与骨髓瘤细胞融合,并按常规单克隆抗体制备技术制备单克隆抗体。 [0178] a cell fusion method: SRRP35 protein immunized animals prepared as described above (including rabbits, goats, etc.), spleen cells were obtained, and then fused with myeloma cells prepared in accordance with conventional techniques for preparing monoclonal antibodies monoclonal antibody.

[0179] b利用噬菌体表面展示库,克隆免疫动物的脾脏IgG可变区并表达成基因工程单克隆抗体。 [0179] b use of phage display libraries cloned animals immunized spleen IgG variable region and expressed as a genetically engineered monoclonal antibody.

[0180] c利用纯化的蛋白免疫动物,制备多抗血清。 [0180] c immunizing an animal with purified protein to prepare polyclonal antiserum.

[0181] 3)检测 [0181] 3) Detection

[0182] a用制备的抗体(多抗或单抗),用组织化学方法进行肝癌的病理检测,阴性信号为肝癌。 [0182] a (multi-resistant or monoclonal) antibody prepared by staining pathological detection of liver cancer, liver cancer negative signal.

[0183] b取患者血清,用ELISA方法检测,阴性反应为肝癌可疑病人。 [0183] b take the serum, detected by ELISA method, a negative reaction to suspected cases of liver cancer.

[0184] c将SRRP35抗体作为蛋白质芯片的探针之一,用于多种肿瘤诊断。 [0184] c will SRRP35 antibody protein probes as one chip for a variety of tumor diagnosis.

[0185] 实施例1:实时定量PCR检测SRRP35在临床样本中的表达 [0185] Example 1: Real-time quantitative PCR SRRP35 expression in clinical samples

[0186] 采用通用方法I~2获得的总mRNA,并通过通用方法4SRRP35在肝癌组织中的表达。 [0186] The general procedure I ~ 2 total mRNA obtained and expressed by the general method 4SRRP35 in hepatocellular carcinoma. 实验中所用检测SRRP35表达的实时定量PCR的引物序列如SEQIDN0.:5和SEQ IDN0.:6所示。 Primer sequences used in the experiment to detect the expression of real-time quantitative PCR SRRP35 as SEQIDN0:. 5 and SEQ IDN0:. Figure 6. 作为内参的β -actin的引物序列如SEQ ID N0.:7和SEQID N0.:8所示。 As the internal control β -actin primer sequence such as SEQ ID N0:. 7 and SEQID N0:. 8 Fig.

[0187] 结果如图1A:在32对临床样本中,通过实时定量PCR检测发现有20对样本肝癌组织中SRRP35的表达明显低于相应的癌旁组织,下调率达到62.5%,统计学分析表明p〈0.01,说明结果的可靠性。 [0187] The results shown in Figure 1A: In the 32 pairs of clinical specimens by real-time quantitative PCR detected 20 samples of liver tissue was significantly lower than SRRP35 noncancerous tissue, reduced rate of 62.5% Statistical analysis showed p <0.01, that the reliability of the results. 同时我们也检测了12种肝癌细胞株中SRRP35的表达模式,如图1B所示,SRRP35在10种细胞中呈现极低的表达,也进一步说明SRRP35在癌中的表达水平较低。 We also examined 12 kinds of human hepatoma cell line SRRP35 expression pattern, as shown in Figure 1B, SRRP35 low expression present in 10 kinds of cells, and further explanation SRRP35 low expression levels in cancer.

[0188] 实施例2:过表达SRRP35基因抑制肝癌细胞的增殖 [0188] Example 2: Overexpression of SRRP35 gene inhibits the proliferation of liver cancer cells

[0189] 以通用方法5、8分别构建真核表达载体,并将SRRP35的cDNA克隆进pcDNA™3.1/myC-His(-)A,转染构建的质粒进入肝癌细胞并成功表达,如图2A所示。 [0189] In the general method of 5,8 respectively eukaryotic expression vector, and SRRP35 the cDNA was cloned into the pcDNA ™ 3.1 / myC-His (-) A, transfected with a plasmid construct into the liver cells and successfully expressed in Figure 2A FIG. 用于构建SRRP35真核表达载体的引物序列如SEQ ID N0.:9 (正向WPSEQ ID N0.: 10 (反向)所示。 Primer sequences used in the construction of eukaryotic expression vector SRRP35 as SEQ ID N0:. 9 (forward WPSEQ ID N0 .: 10 (reverse) below.

[0190] 然后,再以通用方法6~7进行功能实验生长曲线和克隆形成测试,结果如图2B、C所示:SRRP35的过量表达抑制肝癌细胞Sk-h印-1和WRL-68的增殖和克隆形成能力。 [0190] Then, 6-7 in a generic method for functional test growth curve and colony formation test results are shown in 2B, C below: Overexpression SRRP35 inhibit the proliferation of liver cancer cells Sk-h-1 and WRL-68 India's and colony-forming ability. [0191] 实施例3:沉默SRRP35的表达促进细胞生长 3 [0191] Example: Silence SRRP35 expression promoting cell growth

[0192] 用于干扰SRRP35表达的siRNA由上海吉码制药有限公司合成,序列分别如SEQIDN0.: 11 (正义链)和SEQ IDN0.: 12 (反义链)所示。 SiRNA [0192] for interference SRRP35 expressed by the Pharmaceutical Co., Ltd. Hegyi code synthesized sequences are as SEQIDN0 .: 11 (sense strand) and SEQ IDN0 .: 12 (antisense strand) below. 作为干扰对照的NC非特异性核苷酸序列为:SEQIDN0.:13 (正义链)和SEQIDN0.: 14 (反义链) NC as an interference control non-specific nucleotide sequences: SEQIDN0:. 13 (sense strand) and SEQIDN0 .: 14 (antisense strand)

[0193] 经实时定量PCR检测结果如图3A、3B所示:人工合成的干扰siRNA能够有效的下调SRRP35的表达水平,生长曲线实验也证明了SRRP35的下调能够促进肝癌细胞Huh_7和肝癌细胞H印3B的生长。 [0193] by real-time quantitative PCR results in Fig. 3A, 3B shows: synthetic siRNA interference can be effectively reduced the expression level SRRP35, the growth curve experiment also proved Huh_7 and hepatoma cells H printed down SRRP35 can promote liver cancer cells 3B growth.

[0194] 由此可见,SRRP35能够抑制肝癌细胞生长。 [0194] Thus, SRRP35 can inhibit the growth of liver cancer cells.

[0195] 实施例4:SRRP35抑制肝癌细胞的恶性表征 [0195] Example 4: SRRP35 malignancy characterized inhibition of liver cancer cells

[0196] 采用WRL-68作为SRRP35过表达的研究对象,选取了Huh7细胞作为SRRP35沉默表达的的研究对象。 [0196] The WRL-68 as the research object SRRP35 overexpression, select the Huh7 cells as SRRP35 silencing expression study. 通过通用方法9对生长在软琼脂中的细胞克隆数进行分析。 Nine pairs of growth in soft agar cloning cells were analyzed by common methods.

[0197] 结果如图4所示:过表达SRRP35能够有效抑制WRL-68细胞和沉默表达SRRP35能够有效促进Huh-7细胞的恶性程度,表明SRRP35影响肝癌细胞的恶性。 [0197] The results shown in Figure 4: SRRP35 overexpression can suppress WRL-68 cells and silence the expression of SRRP35 can effectively promote the malignant Huh-7 cells, indicating SRRP35 affect malignant hepatoma cells. [0198] 实施例5:SRRP35影响肝癌细胞在裸鼠皮下的成瘤能力 [0198] Example 5: SRRP35 affect liver cell tumorigenicity in nude mice

[0199] 通过通用方法10,将IX IO6个过表达SRRP35的WRL-68细胞、3 X IO6个沉默表达SRRP35的Huh-7细胞和对照细胞分别对称注射入裸鼠腹部皮下,观察瘤体的生长情况。 [0199] by General Method 10, the IX IO6 an overexpression SRRP35 of WRL-68 cells, 3 X IO6 SRRP35 silent expression of Huh-7 cells and control cells were injected into nude mice abdominal subcutaneous symmetry observed tumor growth situation.

[0200] 结果如图5A、5B所示:SRRP35的过表达使肿瘤体积较对照组明显缩小,说明:SRRP35的过表达能有效抑制WRL-68细胞在裸鼠皮下的成瘤能力;而SRRP35的沉默使肿瘤体积明显增大,说明SRRP35的下调有效的促进了Huh-7细胞裸鼠皮下成瘤的能力。 [0200] The results shown in Figure 5A, 5B shown: SRRP35 overexpression of tumor volume was significantly reduced compared with the control group, indicating: SRRP35 overexpression can inhibit WRL-68 cells were tumorigenic in nude mice; and SRRP35 of silence the tumor volume increased significantly, indicating that downward SRRP35 effectively promotes the ability Huh-7 cells subcutaneously into nude mice.

[0201] 在本发明提及的所有文献都在本申请中引用作为参考,就如同每一篇文献被单独引用作为参考那样。 [0201] All documents are referenced in the present invention referred to herein by reference, as if each individual document was cited as a reference. 此外应理解,在阅读了本发明的上述讲授内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。 It should also be understood that, after reading the above teachings of the present invention, those skilled in the art of the present invention that various changes or modifications, these same equivalents as defined by the appended claims fall scope of claims of the present application.

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Referentie
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Juridische gebeurtenissen
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23 juli 2014C06Publication
9 dec 2015C10Entry into substantive examination