转化生长因子β1对人胚肺成纤维细胞结缔组织生长因子及低密度脂蛋白受体相关蛋白表达的影响

目的:探讨体外培养人胚肺成纤维细胞(human pulmonary fibroblasts-1,HPF-1)在外源性转化生长因子β1(transforming growth factor beta, TGF-β1)作用下结缔组织生长因子(connective tissue growth factor, CTGF)与低密度脂蛋白受体相关蛋白(low density lipoprotein receptor related proteins, LRP) 的表达变化及意义.方法:将HPF-1用5 μg/L的TGF-β1分别作用0, 3, 6, 12和 24 h.用RT-PCR方法检测CTGF及LRP mRNA的表达;用蛋白免疫印迹检测LRP蛋白表达变化;用免疫沉淀方法检测TGF-β1作用不同时间,与LRP结合的CTGF的数量变化;用细胞免疫荧光检测细胞中LRP表达情况.结果:相关分析显示,以3 h为关键点,CTGF mRNA(131.53±2.86)与LRP mRNA(224.87±7.00)表达变化具有正相关性(r=0.840 2,P＜0.05);LRP蛋白随TGF-β1作用时间不同,表达水平先升高,于3 h达高峰,而后逐渐下降,用免疫印迹法分析,相对于0 h组(190.85±2.86),3 h组(222.45±3.66)表达具有统计学意义(F=18.06,P＜0.05);用免疫荧光标记细胞LRP也得到类似的结果,比较0 h组(27.56±6.72)和3 h组(88.66±15.72)LRP蛋白表达(F=244.36,P＜0.01)差异具有统计学意义.结论:LRP作为CTGF的受体蛋白,在TGF-β1作用不同时间的情况下,表达量发生变化,并与CTGF的表达成正相关,提示其在肺间质纤维发生中发挥作用.

Expression of connective tissue growth factor and low density lipoprotein receptor related protein induced by transforming growth factor beta 1 in human pulmonary fibroblasts-1

OBJECTIVE: To investigate the tendency and correlation between connective tissue growth factor (CTGF) and low density lipoprotein receptor-related protein (LRP) in human pulmonary fibroblasts-1 (HPF-1) induced by transforming growth factor beta1 (TGF-beta1) at different times. METHODS: After the HPF-1 cells were stimulated with TGF-beta1 (5 microg/L) at different times (0 h, 3 h, 6 h,12 h and 24 h),CTGF and LRP mRNA expressions were analyzed by RT-PCR. The same preparation steps of cell culture were repeated, then, the protein expressions were determined by Western Blot, co-immunoprecipitation Western Blot and immunocytochemistry. RESULTS: The expressions of CTGF and LRP mRNA had similar tendency, and LRP (224.87 +/- 7.00) correlated with CTGF (131.53 +/- 2.86) positively (r = 0.8402, P < 0.05). Furthermore, the protein expression levels had significant difference between 0 h (190.85 +/- 2.86) and 3 h (222.45 +/- 3.66) groups (F = 18.06, P < 0.01),and by immunoprecipitation, it was discovered that the quantity of CTGF bounded to LRP changes, which was similar to the result of LRP western blot. The analysis of immunocytochemistry disclosed the like results as well (3 h: 88.66 +/- 15.72, 0 h: 27.56 +/- 6.72, F = 244.36, P < 0.01). CONCLUSION: The expression of LRP coincides with that of CTGF at the same stimulated time point of TGF-beta1.It suggests that LRP may be involved in the biological process of CTGF. And further research about LRP may provide a new direction to pulmonary fibrosis therapy.