新基因pp3774的亚细胞定位以及在肝癌细胞系和组织中的表达谱研究  

Differential expression study of a novel gene pp3774 in hepatocellular carcinoma cell lines and tissues

作　　者：张锋锐[1] 李锦军[1] 王春艳[1] 葛超[1] 徐文[1] 朱丽[1] 陈燕[2] 万大方[1] 顾健人[1] 

机构地区：[1]上海交通大学肿瘤研究所上海市肿瘤研究所癌基因及相关基因国家重点实验室,上海200032 [2]中科院上海生物工程中心,上海200233

出　　处：《肿瘤》2005年第6期519-524,共6页Tumor

基　　金：国家863项目(编号:2005AA220040;2002AA2Z2002;国家十五攻关重大项目(编号:2003BA711A02-1);上海市科委重大基础研究课题(编号;03DJ14007);上海市卫生局科技发展基金(编号:034012)

摘　　要：目的探讨新基因pp3774在肝癌细胞系、人肝癌及癌旁肝、肝硬化以及正常肝组织中的表达差异及其生物学功能。方法脂质体转染-荧光观察pp3774-GFP融合蛋白的亚细胞定位；合成新基因pp3774特异性多肽,制备兔源性多克隆抗体；用RT-PCR、蛋白印迹、免疫细胞化学及免疫组化方法检测pp3774基因在7个肝癌细胞系及214例肝细胞癌、18例肝硬化及10例正常肝组织中的表达差异。流式细胞术检测转染pp3774基因的SMMC-7721细胞的凋亡率。结果 pp3774蛋白主要定位于细胞质(内质网为主)；RT-PCR检测结果表明pp3774 mRNA在肝癌细胞系BEL-7402、Huh-7、MHCC-97L,及HepG2中高表达,SMMC-7721、MHCC-LM3及人永生化肝细胞系L-02中度表达,而在Hep3B中低表达；肝癌及癌旁组织中pp3774mRNA的表达表现为癌旁高于癌(P<0．05)；免疫组化结果显示pp3774蛋白表达程度依次为正常肝≥肝硬化≥癌旁肝组织>肝癌。转染pp3774基因的SMMC-7721细胞的凋亡发生率高于对照组约10％。结论 pp3774基因是一个具有抑制肝癌细胞生长功能的新基因。Objective To explore the differential expression of a novel gene pp3774 in hepatoma cell lines,human hepatocellular carcinoma and noncancerous liver, cirrhotic liver and normal liver tissues, and its preliminary biological function. Methods Lipofectamine-transfection and fluorescent observation were used to indicate the sub-cellular localization of pp3774-GFP fusion protein. Specific oliopeptide （14 AAs） of pp3774 was synthesized and conjugated to KLH （a vector）, polyclonal rabbit anti-pp3774 antibody was prepared. RT-PCR, western blot, immunocytochemistry, immunohistochemistry were used to detect the differential expression in 7 hepatoma cell lines, 214 HCCs （cancer and noncancerous tissues） , 18 cirrhotic livers and 10 normal livers. FACS was used to analyse the apoptosis in transfected SMMC-7721 cells with pp3774. Results pp3774-GFP fusion protein mainly localized in endoplasmic reticulum of cytoplasm. RT-PCR results showed that pp3774 mRNA highly expressed in BEL- 7402, Huh-7, MHCC-97L and HepG2,moderately expressed in SMMC-7721 ,MHCC-LM3 and immortal human hepatocyte line L-02, and lowly expressed in Hep3B; pp3774 mRNA expression was up-regulated in noncancerous liver tissues compared to HCC tissues（ P d0.05）. Immunohistochemical staining showed that pp3774 protein expression in various tissues had the tendency as follows; normal liver ≥ cirrhotic liver ≥non-cancerous liver〉 HCC tissues. FACS result indicated that pp3774 can induced remarkably apoptosis in SMMC-7721 cell transfected with pp3774 （around 10%）. Conclusion pp3774 gene is a novel candidate with the function of inhibiting hepatoma cell growth.

关 键 词：肝肿瘤 癌 肝细胞 基因pp3774 基因表达 

分 类 号：R735.7[医药卫生—肿瘤]