Yang Miao-Fang, Xie Jun, Gu Xiao-Yi, Zhang Xiao-Hua, Davey Andrew-K, Zhang Shuang-Jie, Wang Ji-Ping, Zhu Ren-Min
Department of Gastroenterology, Jinling Hospital, Second Military Medical University, Zhongshan East Road 305, Nanjing 210002, Jiangsu Province, China.
World J Gastroenterol. 2009 May 7;15(17):2109-15. doi: 10.3748/wjg.15.2109.
To investigate the relationship between 90-kuD ribosomal S6 kinase (p90RSK) and collagen type I expression during the development of hepatic fibrosis in vivo and in vitro.
Rat hepatic fibrosis was induced by intraperitoneal injection of dimethylnitrosamine. The protein expression and cell location of p90RSK and their relationship with collagen type I were determined by co-immunofluoresence and confocal microscopy. Subsequently, RNAi strategy was employed to silence p90RSK mRNA expression in HSC-T6, an activated hepatic stellate cell (HSC) line. The expression of collagen type I in HSC-T6 cells was assessed by Western blotting and real-time polymerase chain reaction. Furthermore, HSCs were transfected with expression vectors or RNAi constructs of p90RSK to increase or decrease the p90RSK expression, then collagen type I promoter activity in the transfected HSCs was examined by reporter assay. Lastly HSC-T6 cells transfected with p90RSK siRNA was treated with or without platelet-derived growth factor (PDGF)-BB at a final concentration of 20 microg/L and the cell growth was determined by MTS conversion.
In fibrotic liver tissues, p90RSK was over-expressed in activated HSCs and had a significant positive correlation with collagen type I levels. In HSC-T6 cells transfected with RNAi targeted to p90RSK, the expression of collagen type I was down-regulated (61.8% in mRNA, P < 0.01, 89.1% in protein, P < 0.01). However, collagen type I promoter activity was not increased with over-expression of p90RSK and not decreased with low expression either, compared with controls in the same cell line (P = 0.076). Furthermore, p90RSK siRNA exerted the inhibition of HSC proliferation, and also abolished the effect of PDGF on the HSC proliferation.
p90RSK is over-expressed in activated HSCs and involved in regulating the abnormal expression of collagen type I through initiating the proliferation of HSCs.
研究90-kD核糖体S6激酶（p90RSK）与体内外肝纤维化发展过程中I型胶原表达之间的关系。
通过腹腔注射二甲基亚硝胺诱导大鼠肝纤维化。采用共免疫荧光和共聚焦显微镜检测p90RSK的蛋白表达和细胞定位及其与I型胶原的关系。随后，运用RNA干扰策略沉默活化的肝星状细胞（HSC）系HSC-T6中p90RSK mRNA的表达。通过蛋白质印迹法和实时聚合酶链反应评估HSC-T6细胞中I型胶原的表达。此外，用p90RSK的表达载体或RNA干扰构建体转染HSC，以增加或降低p90RSK的表达，然后通过报告基因检测法检测转染的HSC中I型胶原启动子的活性。最后，用终浓度为20μg/L的血小板衍生生长因子（PDGF）-BB处理或不处理转染p90RSK siRNA的HSC-T6细胞，并通过MTS转化法测定细胞生长情况。
在纤维化肝组织中，p90RSK在活化的HSC中过表达，且与I型胶原水平呈显著正相关。在转染靶向p90RSK的RNA干扰的HSC-T6细胞中，I型胶原的表达下调（mRNA水平下调61.8%，P<0.01；蛋白质水平下调89.1%，P<0.01）。然而，与同一细胞系中的对照相比，p90RSK过表达时I型胶原启动子活性未增加，低表达时也未降低（P=0.076）。此外，p90RSK siRNA抑制HSC增殖，并且消除了PDGF对HSC增殖的作用。
p90RSK在活化的HSC中过表达，并通过启动HSC增殖参与调节I型胶原的异常表达。
