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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 investigate the expression differences and biological functions of the novel gene pp3774 in hepatocellular carcinoma cell lines, human hepatocellular carcinoma and para-cancerous liver, cirrhosis and normal liver tissues. Methods Lipofectamine was used to observe the subcellular localization of pp3774-GFP fusion protein. A novel pp3774 specific peptide was synthesized to prepare rabbit polyclonal antibody. The expression of pp3774 was detected by RT-PCR, Western blotting, immunocytochemistry and immunohistochemistry Methods The expression of pp3774 gene was detected in seven hepatocellular carcinoma cell lines, 214 hepatocellular carcinoma, 18 cirrhosis and 10 normal liver tissues. Flow cytometry was used to detect the apoptosis rate of pp3774 transfected SMMC-7721 cells. Results The pp3774 protein mainly localized in the cytoplasm (mainly the endoplasmic reticulum). The results of RT-PCR showed that pp3774 mRNA was highly expressed in hepatocellular carcinoma cell lines BEL-7402, Huh-7, MHCC-97L and HepG2, LM3 and human immortalized hepatocyte cell line L-02, while it was low expressed in Hep3B. The expression of pp3774 mRNA in hepatocellular carcinoma tissues and paracancerous tissues was higher than that in carcinoma adjacent tissues (P <0.05). The results of immunohistochemistry Show pp3774 protein expression levels were followed by normal liver ≥ cirrhosis> adjacent liver tissue> liver cancer. The incidence of apoptosis of SMMC-7721 cells transfected with pp3774 gene was about 10% higher than that of the control group. Conclusion The pp3774 gene is a novel gene that inhibits the growth of hepatocellular carcinoma cells.