胰岛素样生长因子结合蛋白相关蛋白1对肝星状细胞转化生长因子β1/Smad3信号通路的影响及意义

The effect of transforming growth factor β1 /Smad3 signaling pathway in hepatic stellate cells induced by insulin-like growth factor binding protein related protein 1

目的探讨胰岛素样生长因子结合蛋白相关蛋白1(IGFBPrP1)对肝星状细胞转化生长因子β1(TGF-β1)/Smad3信号通路的影响及意义。方法体外培养大鼠肝星状细胞株HSC-T6,分别设立空白对照组(加入等量磷酸盐缓冲液)和IGFBPrP1处理组(加入终浓度为30μg/L的IGFBPrP1),作用24 h后收集细胞培养上清液,提取全细胞蛋白,另外收集细胞爬片。培养上清液中TGF-β1的表达采用Western印迹法及ELISA法检测,HSC-T6细胞TGF-β1的表达采用免疫细胞化学法检测;培养上清液中Smad3、p-Smad2/3和纤维连接蛋白(Fn)的表达采用Western印迹法检测。空白对照组与IGFBPrP1处理组TGF-β1、Smad3、p-Smad2/3、Fn蛋白表达量、p-Smad2/3与Smad3蛋白相对表达量比值比较采用独立样本t检验。结果 Western印迹法及ELISA检测结果均显示IGFBPrP1处理组培养上清液TGF-β1蛋白表达量均明显高于空白对照组[0.34±0.06与0.19±0.03相比,(188.34±32.24)ng/L与(136.16±19.52)ng/L相比],免疫细胞化学法也显示HSC-T6细胞TGF-β1蛋白表达量明显高于空白对照组(11.89±0.32与7.71±0.63相比),且差异均有统计学意义(t=3.859,P=0.018;t=3.391,P<0.05;t=14.490,P<0.05);IGFBPrP1处理组与空白对照组HSC-T6细胞中Smad3相对表达量差异无统计学意义(0.65±0.05与0.64±0.05相比,t=0.084,P=0.937),p-Smad2/3蛋白相对表达量、p-Smad2/3与Smad3蛋白相对表达量的比值均明显高于空白对照组,且差异均有统计学意义(0.70±0.05与0.46±0.03相比,t=8.050,P=0.001;1.10±0.12与0.72±0.05相比,t=5.181,P=0.007);IGFBPrP1处理组培养上清液中Fn蛋白相对表达量明显高于空白对照组,且差异有统计学意义(0.52±0.04与0.35±0.02相比,t=5.976,P=0.004)。结论 IGFBPrP1可促进体外培养的HSC-T6产生TGF-β1。IGFBPrP1可使体外培养的HSC-T6中Smad3的磷酸化增加。IGFBPrP1可使体外培养的HSC-T6细胞外基质的重要成分Fn的分泌增加。

Objective To investigate the effect of insulin-like growth factor binding protein related protein 1 （IGFBPrP1） on transforming growth factor [31 （TGF-β1）/Smad3 signaling pathway in hepatic stellate cells. Methods HSC-T6 cells were cultured in vitro and divided into control group （ incubated with equal phosphate buffer saline） and IGFBPrP1 group （ treated with 30 μg/L of IGFBPrP1 ） , then the culture supernatant fluid was collected and whole-cell proteins were extracted and the slides of cells were attained after 24 hours. The expression of TGF-β1 in culture supernatant fluid was detected by both Western blot and ELISA and the expression of TGF-β1in HSC-T6 cells was detected by immunocytochemieal staining; the expressions of Smad3, p-Smad2/3 and fibronectin （Fn） in culture supernatant fluid were detected by Western blot. Comparison between two groups was done with independent sample t-test. Results The results of Western blot and ELISA showed that the expression of TGF-I31 in culture supernatant fluid in IGFBPrP1 group was significantly higher than that in control group [ （0.34 ± 0.06 ） vs （0.19± 0. 03 ）, t = 3. 859, P = 0.018：（188.34 ±32.24） ng/L vs （136. 16± 19.52） ng/L, t=3. 391. P〈0.057. The result of immunocytochemical staining also showed that the expression of TGF-β1 in HSC-T6 cells in IGFBPrPI group was significantly higher than that in control group [ （ 11.89 ± 0.32） vs （7.71 ± 0.63）, t = 14. 490, P 〈 0.05 ]. The difference of the expression of Smad3 in HSC-T6 cells between IGFBPrP1 group and control group was not statistically significant [ （0.65 ± 0.05 ） vs （0.64 ± 0.05 ）, t = 0. 084, P = 0. 937 ]. The expression of p-Smad2/3 and the ratio of p-Smad2/3 and Smad3 in IGFBPrP1 group were higher than those in control group, and the differences were statistically significant [ （ 0.70 ± 0. 05 ） vs （ 0. 46 ± 0. 03 ）, t =8. 050, P=0.001; （1.10＋0.12） vs （0.72＋0.05）, t=5.181, P=0.007]. The expression of Fnin culture supematant fluid in IGFBPrP1 group was significantly higher than that in control group [ （0.52 ± 0. 04） vs （0.35 ~ 0.02）, t = 5. 976, P = 0. 004 ]. Conclusion IGFBPrP1 can promote the production of TGF-β11, increase the phosphorylation of Smad3 in HSC-T6 cells, and increase the secretion of Fn which is the principal component of extracellular matrix of HSC-T6 ceils in vitro.