摘要
目的研究HCV NS3对人肝细胞磷酸化酪氨酸相关蛋白表达的影响。方法通过脂质体稳定转染分别建立表达HCV NS3蛋白的pRcHCNS3/QSG细胞,空白质粒转染细胞pRcCMV/QSG。用Western Blot和免疫细胞化学分别检测磷酸化p44/42,磷酸化p38,磷酸化SAPK/JNK在pRcHCNS3/QSG细胞和pRcCMV/QSG细胞及未转染的QSG7701细胞的表达差异及细胞内的定位;免疫共沉淀实验检测HCV NS3对磷酸化酪氨酸蛋白表达水平的影响。结果pRcHCNS3/QSG细胞,pRcCMV/QSG细胞,未转染的QSG7701细胞抽提的总蛋白中均可检测出磷酸化p44/42,磷酸化p38,磷酸化SAPK/JNK的表达,阳性信号定位于细胞核。上述3种蛋白在pRcHCNS3/QSG细胞的表达较pRcCMV/QSG细胞和未转染的QSG7701细胞表达增强,其磷酸化酪氨酸蛋白的表达也较其他两对照组增强。结论HCV NS3可能通过影响酪氨酸蛋白激酶的表达及活性,使一系列受酪氨酸蛋白激酶调节的蛋白质如MAPK分子异常磷酸化而活化,激活下游信号转导通路,导致肝细胞无限增殖和肿瘤形成。
Objective To investigate the effect of hepatitis c virus NS3 gene on the expression of phosphorylate tyrosine proteins in human hepatocyte. Methods HCV NS3 plasmids and pRcCMV plasmids were stably transfected into QSG7701 cells, which were named as pRcHCNS3/QSG cells and pRcCMV/QSG cells respectively. Western Blot and immunoeytochemistry was used to detect the expression of phospho-p44/ 42, phospho-p38 and phospho-SAPK/JNK in the pRcHCNS3/QSG cells, pRcCMV/QSG cells and QSG7701 cells, respectively. Immunoprecipitation analysis was employed to investigate the effect of HCV NS3 on the phosphotyrosine protein expression. Results The expressions of phospho-p44/42, phospho-p38 and phospho-SAPK/JNK were found in pRcHCNS3/QSG cells, pRcCMV/QSG cells and QSG7701 cells. The positive signals of phospho-p44/42, phospho-p38 and phospho-SAPK/JNK protein were localized in the nucleus of cells. The signals intensity of the pRcHCNS3/QSG cells was stronger than the other the two cells. The expression of phosphotyrosine protein was also up-regulated in pRcHCNS3/QSG cells. Conclusions HCV NS3 may affect cell signaling pathway through activating tyrosine kinase and followed with activation and abnormal phosphorylation of several cytoplasmic signaling molecules such as MAPKs, and induce unlimited hepatocyte transformation and tumor development.
出处
《中国普通外科杂志》
CAS
CSCD
2008年第7期682-686,共5页
China Journal of General Surgery
基金
国家自然科学基金资助项目(30671846)
湖南省自然科学基金资助项目(07JJ4008)
