肝癌相关基因pp1158的表达及功能研究

Expression and function of hepatocel lular carcinoma-related gene pp1158

目的 对pp115 8基因的蛋白表达和体内外功能进行研究。方法 用pET 32a融合表达载体在大肠杆菌BL2 1(DE3)对该基因进行表达 ,制备兔源性多克隆抗体 ;用体外细胞集落形成试验、裸鼠体内成瘤实验、免疫组化及蛋白印迹 ,检测该基因对肿瘤细胞生长的影响及表达差异 ;用Northernblot检测pp115 8在组织中的表达差异。结果 pp115 8对BEL 74 0 2肝癌细胞系的体外集落形成有明显抑制作用 ,抑制率为 5 8.3% (P <0 .0 1) ;对BEL 74 0 2细胞的成瘤有明显抑制作用 (P <0 .0 5 )。蛋白印迹结果显示 ,转染pCMV Script pp115 8的BEL 74 0 2细胞表达pp115 8蛋白。免疫组化结果显示 ,pp115 8基因在人组织中的表达为 :正常肝组织≥癌旁肝组织 >肝癌组织。Northernblot结果显示 ,pp115 8在人胎盘组织中高表达 ,在心、肝、骨骼肌、胰腺、肺等脏器中度表达 ,而在脑和肾组织中表达很低。结论 pp115 8为肝细胞性肝癌的候选抑瘤基因。

Objective To study in vitro and in vivo protein expression and biological function of gen e pp1158, a hepatocellular carcinoma (HCC) related gene. Methods pp1158 was expressed with fusion expression vector pET 32a in E. Coli BL2 1(DE3), and rabbit anti pp1158 fusion protein polyclonal antibody was prepared . The biological function and differential expressions of pp1158 were studied b y in vitro colony formation assay nude mouse in vivo tumor formation assay of transfected BEL7402 cell line and immunohistochemistry and Western blot. Di ffere ntial expression of pp1158 in human fetal tissues were examined by Northern blo t. Results In vitro experiment showed that pp1158 inhibited colony formation rate of transfected BEL 7402 cells, with an inhibition rate of 58.3%( P <0. 01). Tumor formation assay indicated that tumor formation of pCMV Script 1158 tran sfected BEL 7402 cell line was significantly inhibited ( P <0.05) as compared with that of the control group. Immunohistochemical assay showed that pp1158 was expressed in human tissue in the following sequence: normal liver ≥ nonc ance rous liver tissue> HCC. Western blot indicated that a 60kD protein (pp1158 protein) was expressed in BEL 7402 cells transfected with pCMV Script pp1158 DNA, while it was detected in BEL 7402 cells transfected with pCMV Script vector DNA. Northern blot showed pp1158 was expressed in the placenta at a very high level, heart, liver, muscle, pancreas and lung but expressed pcorly in the brain and kidney. Conclusion pp1158 is a new candidate tumor suppressor gene of HCC.