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目的观察糜酶(chymase)对肝星状细胞(HSC)增殖及转分化的影响,探讨糜酶在肝纤维化中的作用。方法用percoll密度梯度离心法分离、培养大鼠HSC,经系列浓度糜酶的作用后,分别进行如下实验:用噻唑比色法(MTT)检测HSC的增殖;Western blot检测细胞内效应蛋白α-SMA及TGF-β1的表达;用RT-PCR法检测TGF-β1及Ⅰ型胶原(Col-Ⅰ)m RNA的表达。结果肥大细胞(MC)糜酶(1、10、100 ng/ml)明显促进HSC增殖,在观察的范围内(1~100 ng/ml)呈质量浓度依赖性,糜酶质量浓度越高,刺激培养的肝星状细胞增殖的能力越强,最高达对照组(无糜酶刺激)的1.45倍(P<0.05);Western blot检测结果显示:糜酶刺激HSC表达α-SMA及TGF-β1明显增强(各实验组与空白对照组比较,P<0.05);RT-PCR法检测HSC上TGF-β1及Col-Ⅰm RNA的表达明显增强,此作用呈剂量依赖性(各实验组与空白对照组比较,P<0.05)。结论肥大细胞糜酶刺激可明显激活HSC,并促进其增殖及转分化为肌成纤维细胞。
Objective To observe the effect of chymase on the proliferation and transdifferentiation of hepatic stellate cells (HSC) and to explore the role of chymase in hepatic fibrosis. Methods The rat HSCs were isolated and cultured by percoll density gradient centrifugation. After the concentration of chymase, the proliferation of HSC was detected by MTT assay. The expression of α- SMA and TGF-β1 were detected by RT-PCR. The mRNA expression of TGF-β1 and Col-Ⅰ was detected by RT-PCR. Results Mast cell (MC) chymase (1, 10, 100 ng / ml) significantly promoted HSC proliferation in a concentration-dependent manner (1-100 ng / ml) in the observed range. The higher chymase concentration and stimulation The ability of cultured hepatic stellate cells to proliferate was up to 1.45 folds higher than that of the control group (chymase stimulated) (P <0.05). The results of Western blot showed that chymase stimulated the expression of α-SMA and TGF-β1 in HSC (All P <0.05). The expression of TGF-β1 and Col-ⅠmRNA in HSC was significantly increased by RT-PCR, which showed a dose-dependent manner (all experimental group and blank control group Comparison, P <0.05). Conclusion The mast cell chymase can activate HSC obviously and promote its proliferation and transdifferentiation into myofibroblasts.