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CN103937871A - Application of SRRP35 gene and expression product thereof to cancer diagnosis and treatment - Google Patents

Application of SRRP35 gene and expression product thereof to cancer diagnosis and treatment Download PDF

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CN103937871A
CN103937871A CN 201310025221 CN201310025221A CN103937871A CN 103937871 A CN103937871 A CN 103937871A CN 201310025221 CN201310025221 CN 201310025221 CN 201310025221 A CN201310025221 A CN 201310025221A CN 103937871 A CN103937871 A CN 103937871A
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gene
cancer
expression
product
treatment
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CN 201310025221
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高勇
李砚东
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上海市东方医院
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K45/00Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K48/00Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Hybridisation probes
    • C12Q1/6883Hybridisation probes for diseases caused by alterations of genetic material
    • C12Q1/6886Hybridisation probes for diseases caused by alterations of genetic material for cancer
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by the preceding groups
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay
    • G01N33/574Immunoassay; Biospecific binding assay for cancer
    • G01N33/57407Specifically defined cancers
    • G01N33/57438Specifically defined cancers of liver, pancreas or kidney
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by the preceding groups
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay
    • G01N33/574Immunoassay; Biospecific binding assay for cancer
    • G01N33/57484Immunoassay; Biospecific binding assay for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57496Immunoassay; Biospecific binding assay for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving intracellular compounds
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/136Screening for pharmacological compounds
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/158Expression markers
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00Screening for compounds of potential therapeutic value
    • G01N2500/10Screening for compounds of potential therapeutic value involving cells

Abstract

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.

Description

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 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 the use SRRP35 gene and expression products in terms of cancer detection, the detection of applications particularly in liver cancer. 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 further relates to SRRP35 genes, proteins and their application in the treatment of cancer agonist.

背景技术 Background technique

[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] hepatocellular carcinoma (hepatocellularcarcinoma, HCC) is our common malignant tumor mortality in second place. 肝癌的诊断,尤其是早期诊断,是临床诊疗和预后的关键。 Diagnosis of liver cancer, 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。 Primary liver cancer can be used as a marker of a biomolecule, generally considered the following four categories: and carcinoembryonic glycoprotein antigen; enzyme and isozymes; cytokine; Gene. 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 (alpha-fetoprotein) mainly, but AFP sensitivity (40% to 65%) and specificity (76% -96%) were unsatisfactory.

[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 have been some local non-surgical treatment options, however, due to the current chemotherapy of liver cancer treatment drugs basically follows other cancers, targeted the poor, Therefore, the poor overall efficacy. [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] Accordingly, there is an urgent need to develop a diagnosis of liver cancer-related protein, in order to effectively inhibit the growth of liver cancer cells, there is an urgent need to develop drugs useful for inhibiting the growth of liver cancer cells, to increase the effectiveness of chemotherapy and specificity .

发明内容 SUMMARY

[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 product SRRP35 applications, especially for the preparation of liver cancer diagnosis and treatment 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] a first aspect the present invention provides the use of a gene or protein SRRP35 SRRP35, detection of cancer for 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, said kit comprising: SRRP35 quantifying mRNA or protein detection reagents 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 agent 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, the above-described agents include detection chip, comprising a nucleic acid chips 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-specific oligonucleotide probe chip comprising a substrate and cancer-related genes spotted on the substrate, said specific oligonucleotide cancer-related genes acid probe comprising a probe that binds to a specific gene or mRNA SRRP35.

[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 specific antibody spotted on the substrate cancer-associated proteins, the cancer-related protein specific antibodies include proteins specific anti SRRP35 antibody.

[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, the fusion protein comprises SRRP35 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 invention there is provided a diagnostic kit for detecting cancer, said kit comprising a container, said container containing a detecting agent for detecting mRNA or protein SRRP35; and a label or instructions the label or instructions specify the kit for the 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 the specification 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 the object to be detected relative SRRP35 β - ratio of expression amounts of mRNA expression of the mRNA of actin relative SRRP35 peritumoral tissue actin ≤ 1, then the prompt detection target is higher than the risk of cancer in 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 chips;

[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 or 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 liver sample.

[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 present invention, there is provided a protein SRRP35, SRRP35 gene or agonist thereof, for the pharmaceutical preparation of cancer cell growth inhibition or proliferation, or manufacture 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] The fourth aspect of the invention there is provided a non-therapeutic method of inhibiting a cancer cell growth or proliferation in vitro, comprising the steps of: at SRRP35 protein or an agonist, 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, the 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 liver cancer 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 screening a candidate compound for treating cancer, 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, the test compound is added at culture system of cells, and the expression was observed in cells of the test group SRRP35 and / or activity; in the control group, the same cell culture system in without adding a test compound and the expression of the cells was observed in the control group SRRP35 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] wherein, if the expression level in the test group SRRP35 cells and / or activity than the control group, it indicates that the test compound is that facilitates the expression and / or activity of SRRP35 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, the 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 step 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) a candidate compound for step (a) is obtained, further testing the inhibition of cancer 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) comprises the steps of: in the test group, the culture system of cancer cells by adding a test compound, and the number of observations and / or growth of cancer cells; in the control group without test compound was added in the culture system of cancer cells, and observe the number and / or growth of cancer cells; wherein, if the test group the number of cancer cells or growth rate than the control group, it indicates that the test compound is cancer proliferation or cell growth inhibition of a candidate compound to treat cancer.

[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 invention there is also provided a method for inhibiting or treating cancer, comprising the steps of: to a subject in need of treatment (mammal) administering a safe and effective amount of a 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, in the range of the present invention, can be combined with each other between the technical features of the invention and in the following technical features (as described in Example) specifically described, thereby constituting 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 to be described herein. 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 of Example 1 Real-time quantitative PCR SRRP35 gene expression of tissue schematic next 32 cases of liver cancer patients cancer tissues and cancer, wherein, "N" refers to adjacent tissues, "C" refers to 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 patients (62.5%) patients with cancer SRRP35 gene expression level is lower than the corresponding adjacent tissue.

[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] FIG. 1B shows the expression pattern SRRP35 real-time PCR detection of genes in the human hepatoma cell line.

[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 case where SRRP35 overexpressed in hepatoma cell line Sk-1fep-1 and WRL-68, illustrating 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 the overexpressed protein SRRP35 weakened colony formation hepatoma cell line Sk-Hep-1 and of WRL-68.

[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 the inhibition of protein expression 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] Figure 3A shows the effective interference of the expression of synthetic genes SRRP35 SiRNA.

[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] Figure 3B shows RNA interference by the method of silencing expression promotes SRRP35 value H and hepatoma cells Huh_7 printed 3B.

[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] FIG. 4 shows a method of silencing expression via RNA interference improves SRRP35 Huh_7 and Hep3B hepatoma cells colony formation in soft agar, described further characterization of malignant hepatoma cell SRRP35 inhibition.

[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 the ability of liver cancer cells WRL-68 form tumors in nude mice SRRP35 overexpression suppression. 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。 Whether SRRP35 overexpression group than the control group on small and light weight and tumor volume, 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 hepatocellular carcinoma cells to 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。 Both in tumor volume and weight than the control group SRRP35 silence the expression of larger and heavier groups, while showing a 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 extensive and in-depth study, for the first time unexpectedly found, SRRP35 low expression in cancer tissues, and tissue beside carcinoma and normal tissue expression, therefore SRRP35 can be used as a marker for cancer detection used to detect or assisted 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。 Further, SRRP35 agonists can inhibit cancer cell growth (in particular liver cancer cells). 在此基础上完成了本发明。 Based on this completed 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 invention also eukaryotic expression vector system is a system-mediated, in hepatoma cell lines Sk-h-1 and printed WRL-68 SRRP35 overexpressed genes (identified as greater than 20 fold over-expression fold). 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 inhibit the growth of hepatoma cells, and Sk-hep-Ι of WRL-68. 因此,可将SRRP35基因用于肝癌的基因治疗。 Thus, SRRP35 genes can be used in gene therapy of liver cancer.

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

[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, refer simply referred to as SRRP35). 应理解,所述术语还包括SRRP35的活性片段和衍生物。 It is understood that the term also includes the active fragments and derivatives thereof SRRP35.

[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, "gene of the invention", "polynucleotide of the invention" refers to a nucleotide sequence encoding a protein or SRRP35 active fragments and derivatives, including sense and antisense nucleic acids. 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", "polypeptide SRRP35" or "cancer marker SRRP35" are used interchangeably and all refer to a protein or polypeptide having the amino acid sequence of human protein 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, known SRSF12 (serine / arginine-rich splicing factorl2, rich in serine and arginine shear factor 12), originally discovered as a result of the SR and a molecular weight 35kD inhibitor 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 of serine-rich and arginine shear factor having RRM RNA binding domain, the regulation of eukaryotic cell having a pre-mRNA transcript to selectively cut off and spliced ​​intron exon functions. 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)。 Found in vivo studies, SRRP35 antagonize SR proteins and activate other adenoviral ElA the pre-mRNA5 'endmost 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 as shown in SEQ IDN0.:1; Genbank Accession Accession No. 135295, cDNA sequences CCDS47459.1 SRRP35 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 a protein such as an amino acid sequence shown in SEQ ID N0.:2.

[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 substance, 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。 The polynucleotides and polypeptides in a natural state in living cells are not isolated and purified, but the same polynucleotide or polypeptide separated from the native state as with other substances present in, 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 to a protein substantially free of naturally SRRP35 associated with other proteins, lipids, carbohydrates or other substances. 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 be purified by 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 capable of producing a single major band on a non-reducing polyacrylamide gel. 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] Polypeptides of the 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 invention may or may not include a starting methionine residue.

[0057] 本发明的多核苷酸可以是DNA形式或RNA形式。 The polynucleotide [0057] The present invention may be in the form of DNA or RNA. DNA形式包括cDNA、基因组DNA或人工合成的DNA。 The form of DNA including cDNA, genomic DNA or synthetic DNA. DNA可以是单链的或是双链的。 DNA may 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。 The mature polypeptide of [0058] SRRP35 encoding comprises: encoding only the mature polypeptide coding sequence; the coding sequence for 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 be a polynucleotide encoding said polypeptide, or may be a polynucleotide comprising a further 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 further relates to variants of the above polynucleotide, polypeptide or polypeptide fragment has the same amino acid sequence, which encodes the analogues and derivatives of the present invention. 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 variant of the polynucleotide may be a variant 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 one or more nucleotide substitutions, deletions or insertions, but does not materially alter the function of its encoded polypeptide.

[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 further relates to a nucleic acid sequence hybridizing with the fragment comprising the sense and antisense nucleic acid fragment. 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 may be used in nucleic acid amplification techniques (e.g. PCR) to determine and / or polynucleotide encoding SRRP35 protein separation.

[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。 Al. [0061] The present invention SRRP35 full length nucleotide sequence or fragments thereof can generally be amplified by PCR method, recombination or synthetic methods available. 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 disclosed, in particular open reading frame sequence to design primers and using a commercially available cDNA library or cDNA library by a conventional method known to those skilled prepared as template, expanded by the related sequences. 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 the sequence is long, often require two or more PCR amplifications, then each time the amplified fragments are 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 is obtained, it may be obtained in large quantities by recombinant methods related sequences. 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 usually cloned into a vector and then into cells, and then isolated from the host cell after the sequences related to proliferation 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, the method can also be used to synthesis of synthetic sequence, especially when fragments of shorter length. 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, by first synthesizing a plurality of small fragments, and then the obtained long fragment 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。 Method [0064] was amplified by PCR DNA / RNA is preferred 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 may be appropriately selected depending on the sequence information of the present invention are disclosed herein, 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。 Standard methods such as gel electrophoresis and purified by the 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 a vector comprising a polynucleotide of the present invention, and host cells with the vector or SRRP35 protein coding sequence of the present invention produced by genetic engineering, and the method of the present invention produce the polypeptide 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 may be used to express or produce recombinant proteins using SRRP35 polynucleotide sequence of the invention. 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) a suitable vector transformed or transduced host cells with a polynucleotide encoding a protein of the present invention SRRP35 (or variant) or by recombinant expression comprising the polynucleotide;

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

[0069] (3).从培养基或细胞中分离、纯化蛋白质。 [0069] (3) isolated from the medium or the 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 well known methods can be used to construct containing human SRRP35 coding DNA sequence and appropriate transcriptional / translational control signals expression vector. 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 techniques, in vivo recombinant techniques, and the like. 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 to an appropriate promoter in the expression vector, 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 further comprises a translation initiation by a ribosome binding site and a 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 dihydrofolate reductase for eukaryotic cell culture use, anti-neomycin as well as green fluorescent protein (GFP), for E. coli, or tetracycline or ampicillin resistance.

[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] containing the appropriate DNA sequence and an appropriate promoter or control sequence, it may be used to transform a suitable host cell so as 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 may be a prokaryotic cell, such as bacterial cells; 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: bacterial cells, E. coli, Streptomyces; fungal cells such as yeast; plant cells; insect Drosophila S2 cells or the Sf9; 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] using conventional techniques well known to those skilled in the host cell transformed with a recombinant DNA. 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, competent cells capable of DNA uptake can be in exponential growth phase were harvested after treatment with CaCl2 method, the steps used are 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 process may be performed using electroporation. 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, optional DNA transfection methods as follows: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposomal 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, expression of the gene of the present invention 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 growing the host cells. 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 selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and the cells 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] The recombinant polypeptide in the above process may be, expressed on the cell membrane or within the cell, or secreted outside the cell. 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 desired, utilizing the physical, chemical and other characteristics of the isolated and purified by various separation methods of recombinant protein. 这些方法是本领域技术人员所熟知的。 These methods are well known to the skilled person. 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 with a protein precipitant (salting out method), centrifugation, osmosis bacteria, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer precipitation, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and methods of these junctions

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 a polyclonal antibody and a monoclonal antibody to human SRRP35 protein, 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 a human SRRP35 gene product or fragment 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, an antibody capable of binding to those SRRP35 human gene products or fragments, but not to recognize and bind other unrelated antigen 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。 Antibody of the invention can be prepared by various techniques known to the person skilled in the art.

[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 includes not only intact monoclonal or polyclonal antibodies, but also antibody fragments having immunological activity, 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] The anti-human antibody SRRP35 protein can be used in immunohistochemical detection of 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] using the protein of the invention, by a variety of conventional screening methods, the substance may be screened SRRP35 interact with proteins, particularly 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] SRRP35 agonist protein of the invention, when administered (administered) in the treatment, can facilitate the expression and / or activity of SRRP35 protein, thereby inhibiting cancer (including hepatocellular carcinoma) 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 generally about 5-8, preferably about pH 6-8, although pH values ​​may vary formulated the nature of the substance and the condition to be treated vary. 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 further provides a pharmaceutical composition comprising a safe and effective amount of a protein or SRRP35 agonist and a pharmaceutically acceptable carrier or excipient according to 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, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. 药物制剂应与给药方式相匹配。 The pharmaceutical formulation 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 invention may be made into the form of injections, for example, prepared with physiological saline or an aqueous solution containing glucose and other adjuvants 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。 Such as tablets and capsules of the pharmaceutical composition, it may 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 dose of the active ingredient is a therapeutically effective amount, e.g. from about I [mu] g -10 mg / kg of body weight per day.

[0085] 检测方法和试剂盒 [0085] Detection methods and kits

[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 further relates to diagnostic tests and quantitative detection of human SRRP35 targeting protein level or mRNA level. 这些试验是本领域所熟知的。 These tests are well 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。 The assay detected SRRP35 human protein, it can be used for diagnosis of 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] if there SRRP35 method for detecting protein in a sample using specific antibodies to detect proteins SRRP35, comprising: contacting the sample with a protein-specific antibodies in contact SRRP35; observing whether antibody complex, the antibody complex is formed SRRP35 it indicates the presence of protein 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 for diagnosis and treatment of diseases associated SRRP35 protein. 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。 Portion of the polynucleotide of the present invention may be used as all or probes are immobilized on a DNA chip or microarray, differential analysis of gene organization and gene expression analysis for 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 protein chips, for SRRP35 protein in the test sample.

[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, comprising specific amplification primer pair of SRRP35 / SRRP35 or specific antibodies.

[0090] 筛选方法 [0090] screening methods

[0091] 本发明还提供了基于SRRP35进行药物筛选的方法。 [0091] The present invention further provides a method of drug screening based on 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。 A method of screening is to affect (promote) the expression or activity of a compound SRRP35 then further tested on cancer compounds screened. 一种筛选方法可基于SRRP35的mRNA的表达水平。 A screening method based on the expression level 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 the representative cancer include (but are not limited to): hepatoma cells.

[0093] 通用方法: [0093] General Procedure:

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

[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 liver cancer patients from surgery, prior to obtaining a sample were signed informed consent and patient. 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一80°C冰箱保存,运输时储存于液氮中。 A liver resection was isolated, excised tumors rapidly than 5cm paraneoplastic tissue surrounding the primary tumor and, frozen in liquid nitrogen and transferred into a refrigerator 80 ° C, 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 were making a final diagnosis by pathologists. 样本依据Edmondson分级标准分为1-1I I级。 Sample is divided into 1-1I I according to the Edmondson grading standards.

[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, the specific operation is as follows:

[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180°C烘箱中烘约4小时,以去除RNA酶; Wash the vessel [0098] I) mortar Pestle homogenizer and the like, respectively, and then DEPC H2O ddH20 rinsed, then baked for about 4 hours at 180 ° C 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) adding the right amount of liquid nitrogen in a mortar to make precooled tissue quickly removed from the liquid nitrogen, cut size about 50-1OOmg, ground to a powder in a mortar;

[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) The curette lapped tissue powder as completely free RNA enzyme moved EP tube, EP tube is previously added to an appropriate volume (Iml) TRIzoI agent, 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,4°C, 12000Xg条件下尚心15分钟; [0101] 4) at room temperature for 5 minutes, chloroform was added proportion (200μ 1 / lml TRIzol) to the centrifuge tube, shaken vigorously rapidly for 15 seconds, allowed to stand at room temperature for 2-3 minutes, 4 ° C, still under the conditions of the heart 15 12000Xg minute;

[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,4°C,12000 X g条件下离心10分钟,此时可见RNA沉淀; [0102] 5) The upper aqueous phase as possible to a new EP tube-free RNA enzyme, an equal volume of isopropanol, mix by inverting 5 times and allowed to stand at room temperature 10 minutes, 4 ° C, 12000 X g rpm for 10 minutes under conditions of RNA pellet was seen at this time;

[0103] 6)将上清倒掉,加入75%乙醇(lml/lml TRIzol),混匀,洗涤RNA,离心4°C,7500 X g条件下离心5分钟; [0103] 6) The supernatant was discarded, and added 75% ethanol (lml / lml TRIzol), mixing, centrifugation and washed under an RNA, centrifugation 4 ° C, 7500 X g for 5 minutes;

[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, the precipitate was dried natural 5-10min (careful not to completely dry); added 30 - 50 μ I DEPC H2O, pipetting several times, the RNA pellet was dissolved;

[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,一80°C保存。 [0105] 8) The RNA concentration and purity then determined 0D260 / 280 (1.8 - 2.0); gel electrophoresis degrade absence, a 80 ° C storage.

[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] RNA extraction cell line, cells in logarithmic growth phase, the culture was drawn, corresponding amount of TRIzol reagent (lml TRIzol / lOcm2) based on the area of ​​the petri dish cells were lysed by pipetting a few times, the cells will be cleaved EP tube to collect the RNA-free enzyme remaining chloroform completed according to the above steps 4) - 8) - isopropanol were isolated 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] with 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) the following components were added in the EP in nuclease-free tube:

[0110] [0110]

Figure CN103937871AD00101

[0111] 置于PCR仪中,70°C,5分钟,然后立即在冰上冷却5min。 [0111] placed in PCR machine, 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-described system was added the following components:

[0113] [0113]

Figure CN103937871AD00102

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

[0115] 逆转得到的cDNA置于4°C保存。 [0115] cDNA obtained reversal 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 reaction using SYBR * Premix ExTaq ™ (Perfect RealTime) kit (TaKaRaBiotechnology C0., Ltd.Dalian, China) reaction system, using Thermal CyclerDice ™ RealTime System (TP800 real-time PCR instrument, of 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 amplification product length to 80bp - 150bp most appropriate (can be extended to 300bp).

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

[0119] [0119]

Figure CN103937871AD00103

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

[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 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 [0126] the fluorescent background signal and the threshold value the default value setting of the instrument, will be automatically generated after each PCR reaction, Ct value represents the fluorescence signal in each reaction tube reaches a set threshold value (10 times the fluorescence intensity of baseline) of the number of cycles experienced; SRRP35 gene of each inner tube 3 do template, Ct values ​​obtained were averaged; Ct by subtracting the average Ct average SRRP35 gene template corresponding reference gene (β-actin) to give 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 group subtracted ACt HCC corresponding adjacent tissues to obtain Λ Λ Ct value, the next multiple of SRRP35 gene hepatocellular carcinoma and the group treated with 2 _ "^ table

/Jn ο / Jn ο

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

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

[0129] 2)真核表达载体的选择:pcDNA™3.1/myc-His (-) A, 5522nucleotides。 [0129] 2) Select adenovirus vector: 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, a binding expression vector pcDNA ™ 3.1 / myc-His (-) restriction sites A primer design, primer sequences SEQ ID N0.:3 (forward) and SEQIDN0.:4 (reverse) 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 in SRRP35 stop codon is removed, so that the band of the C-terminal SRRP35 6xHi s and c-myc tags. 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: Using a high fidelity DNA polymerase PrimeSTAR ™ HS DNA Polymerase (TaKaRa), to amplify the gene SRRP35 L02cDNA full-length open reading frame as a template, 50 μ I of the total composition of the reaction system as follows:

[0131] [0131]

Figure CN103937871AD00111

[0132] 采用两步PCR法(98 °C,IOsec ;60°C,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 PCR product size of about

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 size, tapping recovered (gel purification kit: MACHEREY-NAGEL) conform to the size of the PCR product fragment.

[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) and digested PCR product was recovered plasmid pcDNA ™ 3.1 / myc-His (-) A, following digestion reaction:

Figure CN103937871AD00112

[0135] 37°C酶切反应I小时;割胶回收酶切产物。 [0135] 37 ° C h I digestion reaction; tapping cleavage product recovered. [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 1,16°C连接2h乃至过夜; [0136] 4) is connected: the PCR product was digested with the carrier recovered according molar ratio (4: 1 mixing ratio) is, the 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,42°C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,37°C、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,37°C培养12 - 16小时。 [0137] 5) Conversion: Take 10 μ I ligation 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 30min, so that recovery and amplification generation cells, centrifuged 3000rpm 2min, most of the supernatant was removed, leaving 50 - 100 μ I gently pipetting bacteria ,, mixing the precipitate, and then evenly applied onto LB plates have ampicillin resistance (Amp +) of, 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) clones were identified: the picked ampicillin-resistant colonies were screened after growth in liquid media plus expanded ampicillin, the plasmid identified by restriction enzyme extraction: Take 1- 2 μ g plasmid miniprep using the EcoRV, Hind III double digestion, agarose gel electrophoresis to identify restriction fragments of size, vector pcDNA ™ 3.1 / myc-His (_) a fragment of a size of about 5.5kb, SRRP35 reading frame of about 800bp fragment size, in line with the clone size to send sequencing confirm the correctness of the insertion 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 [0140] I) the different types of HCC cells is calculated by the total number of cells 3-5X 103/100 μ I / aperture according to their growth characteristics, fully digested 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; Day for three wells each, press 5--7 cells seeded on day;

[0141] 2)待细胞基本贴壁后观察细胞状态和数目。 [0141] 2) to be substantially adherent cells and observe the state of cell number. 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-8,37°C,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 developer (CellCountingKit-8, D0JIND0, Japan), per 100 μ I medium plus 10 μ I CCK-8,37 ° C, 5% C02 incubator disposed incubated lh, ELISA instrument absorbance measured at 450nm, recording, determining the actual starting cell density, as a 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) per day or half the amount was changed the next day, depending on the experiment may request;

[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; Cell morphology was observed [0143] 4) microscope, a fixed time interval measurement, recording cell growth conditions;

[0144] 5) 一般测5至7天。 [0144] 5) measured generally 5 to 7 days. 待测定结束后,收集数据进行处理,用Excel绘出图表。 After completion of the measurement, the data collection process, plotted using Excel charts.

[0145] (7)细胞克隆形成实验 [0145] (7) cells in clonogenic 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: using Lipofectamine ™ 2000 (Invitrogen) transfected cells, overexpression or silencing expression SRRP35 gene in a cell;

[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) The cells were transfected in 6-well plates or a 35mm petri dish was digested with normal culture medium 24h after counting a certain number of vaccination to IOOmm dish (a different number of different cell lines), cultured for 24h, and then Add appropriate concentration of cells based on the type 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 every 3-5 days and replaced with fresh medium plus G418 selection until a visible 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 solution in the petri dish, IXPBS washed twice with Coomassie Brilliant Blue R-250 staining 2h, and gently rinsed with water, and then stained with Coomassie 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 photographs, according to the same standard (the size of cell clones) cell clones were counted on each dish.

[0151] (8)蛋白质印迹(Western Blot) [0151] (8) Western blot (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,一80°C保存; [0152] I) Preparation of protein samples: after withdrawing the cultured cells to the culture supernatant, were washed twice with IXPBS-cold lysis buffer was added 2X SDS (IOOmM Tri s_Cl, pH = 6.8,4% SDS, 20% glycerol) after cleavage sufficiently, heated in boiling water bath IOmin, 12000 Xg centrifugation IOmin, supernatant was transferred to a new tube, Pierce 'BCA ProteinAssayKit obtained protein was quantified, a 80 ° C storage;

[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: Protein samples were added in an appropriate amount of the sample containing 200mM DTT buffer (loadingbuffer), heated in boiling water bath 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 film: The electrophoretic 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 ; ; Put the order of the negative electrode, wet gyroscope (XCellSureLock ™, invitrogen) 30 volts wiped film 30_40min - _ the I layer thickness of the positive electrode plate filter _ _ nitrocellulose membrane filter electrophoretic gel thin plate by -2

[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 /0.P/oPBST as a blocking solution, a 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4°C孵育过夜,0.1%PBST洗三次,每次5min ; [0156] 5) an antibody: antibody diluted in blocking buffer (recommended specification reference antibody concentration), incubated 2h incubation at room temperature or 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) a secondary antibody: fluorescent secondary antibodies diluted in blocking buffer (1: 1000) and 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) scavenging film: 0DYSSEY scanning infrared imaging system 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) Make 1% and 2% low-melting Agarose (TaKaRa company), 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 medium (2.5 X DMEM, containing 20% ​​FBS);

[0162] 3)将37°C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4°C冰箱中,待凝固后使用; [0162] 3) The 37 ° C and incubated in 2% Agarose 2XDMEM culture was mixed in the same volume, 0.5ml per well was added to 24-well plates, placed in 4 ° C refrigerator, used to be solidified;

[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 digested into a single cell culture cells, counted, diluted to the same concentration (0.5ml of 3000-5000);

[0164] 5)将细胞悬液与37°C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4°C冰箱IOmin ; [0164] 5) The cell suspension was incubated with 37 ° C in l% Agar0Se same volume mixed, added to 24-well plates have been laid in the lower layer of plastic, 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 0.2ml soft agar solidified culture medium and placed in 37 ° square, 0 5%) incubator was continued 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 [0166] 7) microscope, counted, analyzed.

[0167] (10)裸鼠成瘤实验 [0167] (10) nude mice Experiment

[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) The mice used were 5-6 weeks old male BLAB / c nu nude mice, provided by Shanghai SLAC Limited experimental animals, housed in animal models Culture Center 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) treated cells take, with the same number of mice inoculated subcutaneously (different number of different types of cells seeded), in order to avoid errors caused by individual differences, cells treated with different symmetrical same mice inoculated ;

[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, After [0170] 3) to be visible tumors appeared, tumor size monitored every three days, caliper reading long and short diameters of the tumor,

[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 by the following formula: volume = long diameter short diameter 2 X;

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

[0173] (11)抗体获得和免疫检测 [0173] (11) an antibody obtained and immunodetection

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

[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 a human cDNA sequence database, Genebank SRRP35 gene coding block obtained by the PCR amplification, inserting a prokaryotic or eukaryotic vector, protein expression SRRP35 press genetically engineered to express the product purified protein purification systems.

[0176] 2)抗体制备[0177] 可采用以下几种方法制备抗体: [0176] 2) Preparation of Antibodies [0177] Antibodies can be prepared using the following methods:

[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: immunizing an animal with a protein SRRP35 prepared above (including rabbits, goats, etc.), spleen cells were obtained, and then fused with myeloma cells, according to conventional monoclonal antibody preparation techniques preparing monoclonal antibodies.

[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 of a phage display library, the variable region cloned spleen IgG of the immunized animal 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, prepared 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 (polyclonal or monoclonal) antibodies prepared, for pathological examination of liver cancer by histochemical methods, a negative signal liver cancer.

[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 taken serum, by ELISA method, liver cancer patients suspected negative reaction.

[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 antibody as one of the probes SRRP35 protein chips for the diagnosis of a variety of tumors.

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

[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] I ~ total mRNA obtained using general method 2, 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。 Used in the assay detected the expression of SRRP35 primer sequences are shown in real-time PCR and SEQIDN0.:5 SEQ IDN0.: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 an internal control β -actin primer sequence as shown in SEQ ID N0.:7 and SEQID N0.:8.

[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] Results in FIG. 1A: 32 clinical samples by real-time quantitative PCR showed expression SRRP35 20 HCC samples was significantly lower than the corresponding adjacent tissue, reduction rate of 62.5% Statistical analysis indicated 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 the expression pattern of 12 kinds SRRP35 hepatoma cell lines, as shown in FIG. 1B, SRRP35 exhibit extremely low expression in 10 kinds of cells, and further described in a lower level of expression SRRP35 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 SRRP35 gene inhibits proliferation of hepatoma 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 General Method 5, 8 were constructed eukaryotic expression vector and cDNA SRRP35 cloned into pcDNA ™ 3.1 / myC-His (-) A, into the plasmid construct transfected and successfully expressed in hepatoma cells, as shown in FIG 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。 The primer sequences used to construct expression vectors SRRP35 true as SEQ ID N0.:9 (forward WPSEQ ID N0 .: 10 (reverse) FIG.

[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, to the general method of function experiments 6-7 growth curve and colony formation test, the results in Figure 2B, as shown in C: overexpression SRRP35 inhibit the proliferation of hepatoma cells Sk-h-1, and printed in WRL-68 colony formation and. [0191] 实施例3:沉默SRRP35的表达促进细胞生长 3 [0191] Example: silencing the expression promotes cell growth SRRP35

[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。 Expression of siRNA SRRP35 [0192] interference caused by a code Pharmaceutical Co. Hegyi synthesized sequences as SEQIDN0 .: 11 (sense strand) and SEQ IDN0 .: 12 (antisense strand) shown in FIG. 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) As a non-specific nucleotide sequence to control interference NC: 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] Real-time quantitative PCR detection result by FIG. 3A, FIG 3B: synthetic siRNA interference can be effectively decreased the expression of SRRP35 growth curve experiment also proved capable of promoting SRRP35 downregulation of liver cancer cells and liver cancer cells H printed Huh_7 growth 3B.

[0194] 由此可见,SRRP35能够抑制肝癌细胞生长。 [0194] Thus, SRRP35 capable of inhibiting the growth of hepatoma cells.

[0195] 实施例4:SRRP35抑制肝癌细胞的恶性表征 [0195] Example 4: SRRP35 malignant cells characterized by inhibition of hepatoma

[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] WRL-68 employed for the study SRRP35 overexpressed, Huh7 cells were selected for the study of the expression silencing SRRP35. 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。 Log cell clonal growth in soft agar was analyzed by General Method 9.

[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: Overexpression SRRP35 WRL-68 effective to inhibit expression of malignant cells and silence can effectively promote malignant SRRP35 Huh-7 cells, indicating that the influence SRRP35 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 tumorigenicity in nude mice hepatoma cells

[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 overexpressed SRRP35 of WRL-68 cells, 3 X IO6 SRRP35 silent expression of Huh-7 cells and control cells were injected into nude mice symmetric abdominal subcutaneous, tumor growth was observed Happening.

[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 in FIG. 5A, 5B shown: overexpressing tumor volume was significantly reduced compared with the control group, indicating the SRRP35: SRRP35 overexpression can inhibit tumorigenicity in nude mice of WRL-68 cells; and the SRRP35 silencing the tumor volume increased significantly, indicating SRRP35 downregulation effectively promote the ability of 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] The present application are incorporated in all documents mentioned herein incorporated by reference, as if each reference were individually incorporated by reference above. 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。 Furthermore, it should be understood that, after reading the above teachings of the present invention, those skilled in the art that various changes or modifications may be made to the present invention, and these equivalents also fall within the present application as defined in the appended claims scope.

Claims (10)

  1. 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. 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 according to claim 1, wherein, said kit comprising: SRRP35 quantifying mRNA or protein detection reagents and the corresponding label or instructions.
  3. 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 1, wherein said reagent comprises SRRP35 specific primers, specific antibodies, probes and / or chips; cancer and / or liver cancer comprising.
  4. 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 the kit comprises a container, said container containing a detecting agent for detecting mRNA or protein SRRP35; and a label or instructions, the label or instructions indicate the kit for detecting cancer.
  5. 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 specification indicate the following: When the object to be detected relative SRRP35 β - actin mRNA expression in cancer tissues adjacent opposite SRRP35 β - the amount of mRNA expression of actin ratio ≤1, then the chances of prompt detection target cancer than the general population.
  6. 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, gene or agonist SRRP35 use, characterized in that the medicament is used for the preparation of cancer cell growth inhibition or proliferation, or manufacture of a medicament for the treatment of cancer.
  7. 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.- A method of treating seed inhibiting cancer cell growth in vitro or non-proliferation, characterized by comprising the steps of: at SRRP35 protein or an agonist, cultured cancer cells, thereby inhibiting cancer cell growth or proliferation.
  8. 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 treating cancer, characterized in that, said method comprising the steps of: (a) the test group, the test compound is added at culture system of cells, and the cells were observed in the test group SRRP35 the expression and / or activity; in the control group, was not added in the same cell culture system a test compound and the expression of the cells was observed in the control group SRRP35 and / or activity; wherein if the cells in test group SRRP35 expression level and / or activity than the control group, it indicates that the test compound is SRRP35 expression and / or activity of promoting effect of a candidate compound for treating cancer.
  9. 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, wherein said method further comprises the step of: (b) a candidate compound for step (a) is obtained, further testing inhibitory effect on cell growth or proliferation.
  10. 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) comprises the steps of: in the test group, the culture system of cancer cells by adding a test compound, and the observed number of cancer cells and / or growth ; in the control group, the test compound was not added in the culture system of cancer cells, and observe the number and / or growth of cancer cells; wherein, if the test group the number of cancer cells or growth rate than the control group, it indicates that the test compound is a candidate compound to inhibit cancer cell growth or proliferation of cancer treatment.
CN 201310025221 2013-01-23 2013-01-23 Application of SRRP35 gene and expression product thereof to cancer diagnosis and treatment CN103937871A (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2200645A1 (en) * 2007-10-10 2010-06-30 The Trustees of Princeton University Cytomegalovirus vaccines and methods of production
EP2302036A2 (en) * 2005-05-27 2011-03-30 Lifescan, Inc. Amniotic fluid derived cells
WO2012087983A1 (en) * 2010-12-20 2012-06-28 The General Hospital Corporation Polycomb-associated non-coding rnas

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2302036A2 (en) * 2005-05-27 2011-03-30 Lifescan, Inc. Amniotic fluid derived cells
EP2200645A1 (en) * 2007-10-10 2010-06-30 The Trustees of Princeton University Cytomegalovirus vaccines and methods of production
WO2012087983A1 (en) * 2010-12-20 2012-06-28 The General Hospital Corporation Polycomb-associated non-coding rnas

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Y. LI ET AL.: "SRrp35 suppresses cell proliferation and malignancy in hepatocellular carcinoma", 《HEPATOLOGY RESEARCH》 *

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