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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 the protein expression of pp115 8 gene in vitro and in vivo. Methods The gene was expressed in Escherichia coli BL21 (DE3) by using pET32a fusion expression vector and the rabbit polyclonal antibody was prepared. The in vitro cell colony formation assay, tumorigenicity test in nude mice, immunohistochemistry and Western blotting were performed. The effect of the gene on the growth of tumor cells and the difference of the expression were detected. The differences of the expression of pp115 8 in the tissues were detected by Northern blot. Results pp115 8 had a significant inhibitory effect on colony formation of BEL 74 0 2 hepatocellular carcinoma cell lines in vitro with a inhibitory rate of 5 8.3% (P <0.01) and a significant inhibition of tumorigenesis in BEL 74 0 2 cells (P <0 .0 5). Western blot results showed that pp1158 protein was expressed in BEL-7402 cells transfected with pCMV Script pp115 8. The results of immunohistochemistry showed that the expression of pp115 8 gene in human tissues was: normal liver tissue> paracancer liver tissue> liver cancer tissue. Northern blot results showed that pp115 8 was overexpressed in human placenta and moderately expressed in heart, liver, skeletal muscle, pancreas, lung and other organs, but was low in brain and kidney tissues. Conclusion pp115 8 is a candidate tumor suppressor gene for hepatocellular carcinoma.