大、小剂量TGF-β1刺激大鼠皮肤成纤维细胞24h后检测c-Ski和Smad3的表达及其生物学意义

Expression of c-Ski and Smad3 in skin fibroblast of rat at 24 h after induction by the high or low concentration of TGF-β1

目的:探讨大、小剂量转化生长因子β1(TGF-β1)刺激大鼠皮肤成纤维细胞24h后检测c-Ski和Smad3的表达及其生物学意义.方法:大、小剂量TGF-β1刺激体外培养的大鼠皮肤成纤维细胞,24h后采用WesternBlot和荧光实时定量PCR观察c-Ski,Smad3,CyclinD蛋白和c-Ski,Smad3mR-NA水平的变化情况;pcDNA3.0-LacZ报告基因免疫组化染色来确定转染效率,BrdUELISA法检测c-Ski和Smad3质粒分别转染或转染同时加大、小剂量TGF-β1刺激后细胞增殖的变化.结果:不同剂量TGF-β1刺激24h后,小剂量刺激可显著提高c-Ski,CyclinD的表达水平,而Smad3表达水平无显著变化;大剂量刺激可显著提高Smad3的表达水平和显著降低CyclinD的表达水平,c-Ski表达水平无显著变化.转染c-Ski可以促进细胞增殖,但同时降低了大、小剂量TGF-β1调节细胞双向增殖的幅度;转染Smad3可以抑制细胞增殖同时也降低了大、小剂量TGF-β1调节细胞双向增殖的幅度.结论:大、小剂量TGF-β1刺激24h后引起Smad3和c-Ski表达不同变化决定了细胞的增殖状态,这也是大、小剂量TGF-β1双向调节细胞增殖作用的可能机制之一.

AIM：To study the significance of c-Ski and Smad3 expression in skin fibroblast of rat at 24 h after induction by the high or low concentration of TGF-β1 in vitro.METHODS：The protein expression of c-Ski,Smad3,Cyclin D and mRNA expression of c-Ski,Smad3 were examined by Western Blot and Real-time PCR assays respectively.The transfection efficiency of the pcDNA3.0 vector was measured by β-gal method.Cell proliferation was determined by the measurement of BrdU incorporation into newly synthesized cellular DNA using a cell proliferation ELISA kit（Roche） induced by the high or low concentration of TGF-β1 with or without transfection of c-Ski plasmid and Smad3 plasmid respectively.RESULTS：The protein and mRNA expression of c-Ski and Smad3 were significantly increased at the low and at the high concentration of TGF-β1 respectively.The protein expression of Cyclin D was increased at the low concentration of TGF-β1,but decreased at the high concentration of TGF-β1.Moreover,c-Ski overexpression promoted fibroblast proliferation and abrogated bi-directional role of the high or the low concentration of TGF-β1 on fibroblast proliferation,and Smad3 overexpression inhibited fibroblast proliferation and abrogated bi-directional role of the high or the low concentration of TGF-β1 on fibroblast proliferation.CONCLUSION：The c-Ski and Smad3 expression different change at 24 h after high or low TGF-β1 concentration in turn determines the proliferation role of fibroblast,and suggests an important role of c-Ski and Smad3 in the promoting or inhibiting effects of TGF-β1 on fibroblast proliferation.