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目的研究糜酶对肝星状细胞转化生长因子β1(TGF-β1)/Smad3信号通路的影响。方法对大鼠肝星状细胞株HSC-T6进行体外培养,设定2组,空白对照组(加入等量磷酸盐缓冲液)及糜酶处理组(加入终浓度为10mg/L的糜酶),作用24h后收集细胞培养上清液,提取全细胞蛋白,收集细胞爬片。TGF-β1在培养上清液中的表达采用免疫印迹法及ELISA法检测,Smad3、p-Smad2/3、纤维连接蛋白(Fn)的表达采用免疫印迹法检测。结果免疫印迹法及ELISA检侧结果均表明在糜酶处理组培养上清液中TGF-β1蛋白表达量均较空白对照组高[免疫印迹法:(0.33±0.05)vs(0.18±0.03),t=3.826,P<0.05;ELISA法:(178.05±34.63)ng/L vs(138.42±20.17)ng/L,t=3.401,P<0.05],差异均有统计学意义;而糜酶处理组与空白对照组在培养上清液中Smad3相对表达量比较差异无统计学意义[(0.65±0.06)vs(0.62±0.05),t=0.086,P=0.891>0.05];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则明显高于空白对照组,差异均有统计学意义[(0.72±0.04)vs(0.45±0.03),t=8.101,P=0.001;(1.11±0.13)vs(0.70±0.04),t=5.210,P=0.006<0.05];糜酶处理组Fn的表达量较空白对照组明显升高,差异有统计学意义[(0.56±0.04)vs(0.34±0.02),t=6.141,P<0.05]。结论对体外培养的HSC-T6,糜酶可促进其产生TGF-β1,并使其中的Smad3的磷酸化增加。在糜酶作用下HSC-T6的细胞外基质成分Fn分泌明显增加。
Objective To investigate the effect of chymase on the expression of TGF-β1 / Smad3 in hepatic stellate cells. Methods Rat hepatic stellate cell line HSC-T6 was cultured in vitro. Two groups were set up, the blank control group (adding the same amount of phosphate buffer) and the chymase treatment group (chymase adding the final concentration of 10 mg / L) After 24 hours, the cell culture supernatant was collected, the whole cell protein was extracted, and the cell slide was collected. The expression of TGF-β1 in culture supernatant was detected by Western blotting and ELISA. The expression of Smad3, p-Smad2 / 3 and fibronectin (Fn) were detected by Western blotting. Results The results of Western blotting and ELISA showed that the expression of TGF-β1 in the culture supernatant of chymase group was higher than that of the blank control group (Western blotting: 0.33 ± 0.05 vs 0.18 ± 0.03, t = 3.826, P <0.05; ELISA method: (178.05 ± 34.63) ng / L vs (138.42 ± 20.17) ng / L, t = 3.401, P <0.05] .The difference was statistically significant Compared with the blank control group, the relative expression of Smad3 in the culture supernatant had no significant difference ([(0.65 ± 0.06) vs (0.62 ± 0.05), t = 0.086, P = 0.891> 0.05] (0.72 ± 0.04) vs (0.45 ± 0.03), t = 8.101, P = 0.003, P = 0.031, P = (1.11 ± 0.13) vs (0.70 ± 0.04), t = 5.210, P = 0.006 <0.05]. The expression of Fn in chymase group was significantly higher than that in control group [(0.56 ± 0.04) vs (0.34 ± 0.02), t = 6.141, P <0.05]. Conclusion HSC-T6 chymase can promote the production of TGF-β1 and increase the phosphorylation of Smad3 in vitro. Chymase under the action of HSC-T6 extracellular matrix components Fn secretion increased significantly.