中文摘要：

目的探讨血管紧张素Ⅱ（AngⅡ）促进肝星状细胞（HSC）收缩时Rho激酶介导的非Ca^2＋依赖性信号转导通路的作用机制。方法采用HSC—T6细胞系，给予AngⅡ10μmol／L处理，聚硅酮膜法直观检测HSC的收缩性；蛋白质印迹法检测肌球蛋白轻链（MLC）和磷酸化MLC表达水平。观察AngⅡ1型受体阻断剂伊贝沙坦、蛋白激酶C特异性抑制剂stauro、Rho激酶特异性抑制剂Y27632、肌球蛋白轻链激酶特异性抑制剂ML-7对磷酸化MLC表达水平的影响；逆转录聚合酶链反应检测Rho-Rock通路RhoA激酶2（Rock2）、RhoAGTP、Rho鸟核苷酸转化因子（RhoGEF）mRNA的表达。结果AngⅡ可诱导HSC收缩；还可诱导磷酸化MLC蛋白水平的变化，并呈时间依赖性，15min达到峰值后逐渐减低。AngⅡ＋伊贝沙坦组和AngⅡ＋Y27632组诱导的MLC蛋白磷酸化水平均低于AngⅡ组（1．12±0．09、1．22±0．10vs1．33±0．06，P=0．003）；而AngⅡ＋ML-7＋stauro组磷酸化MLC蛋白水平（1．43±0．09）高于AngⅡ＋Y27632组（P=0．003）；AngⅡ＋Y27632＋ML-7＋stauro组水平较低（0．64±0．04，P=0．000）。AngⅡ组Rock2、RhoAGTP、RhoGEF mRNA的表达均高于相应对照组（0．36±0．01vs0．12±0．01、0．80±0．01vs0．40±0．02、0．65±0．11vs0．33±0．09，均P〈0．05），AngⅡ＋伊贝沙坦组3种元件的表达均低于AngⅡ组。AngⅡ＋Y27632组Rock2与RhoGEF mRNA的表达（0．15±0．01、0．28±0．08）较AngⅡ组低，RhoA GTP的表达（1．14±0．02）则较高。AngⅡ＋ML-7＋stauro组3种元件的表达均高于对照组，AngⅡ＋Y27632＋ML-7＋stauro3组3种元件的表达（0．23±0．01、0．83±0．02、0．69±0．08）均高于AngⅡ＋ML-7＋stauro组（均P〈0．05）。结论AngⅡ可以通过Rho—Rock通路来诱导HSC的收缩。

英文摘要：

Objective To investigate the mechanism of Ca^2＋ -independent pathways mediated by Rho-kinase in contraction of hepatic stellate cells （HSCs） induced by angiotonin Ⅱ （ Ang Ⅱ ）. Methods Human HSCs of the line HSC-T6 were cultured and randomly divided into 6 groups ： negative control group, Ang Ⅱ group treated by Ang Ⅱ 10 μmol/L for 15 min, Ang Ⅱ ＋ irbesantan （Ang Ⅱ receptor inhibitor） group, exposed to irbesantan for 60 min prior to Ang Ⅱ treatment, Ang Ⅱ ＋ Y27632 （ Rho kinase specific inhibitor） exposed to Y27632 for 60 min prior to Ang Ⅱ treatment, Ang Ⅱ ＋ ML-7 （ myosin light chain kinase specific inhibitor） ＋ saturo （ protein kinase C specific inhibitor） group exposed to stauro for 60 min prior to Ang Ⅱ treatment, and Ang Ⅱ ＋ Y27632 ＋ ML-7 ＋ stauro group, exposed to Y27632 and stauro for 60 min prior to Ang Ⅱ treatment. The cell contraction was detected by silicone-rubber-membrane cultivation directly. The protein levels of MLC and phosphorylated MLC were detected by Western blotting 5, 15, 30, 60, and 120 rain after Ang Ⅱ treatment. RT-PCR was used to detect the expression of Rock2, RhoAGTP, and RhoGEF in Ca^2＋ independent pathways mediated by Rho-kinase. Results The silicone-rubber-membrane covered by Ang Ⅱ treated HSCs showed obvious wrinkles indicating the contraction of HSCs. The ratios of phosphorylated MLC protein at the time pints 5, 15, 30, 60, andl20 min of the Ang Ⅱ group to the control group （Omin）were 11.7 ±0.1, 26.9 ±0.1, 11.2 ±0.1, 4.1 ±0.1, and 1.0 ±0.1, showing that Ang Ⅱ increased the phosphorylated MLC protein level time-dependently with the peak level at the time point of 15 minutes. The levels of phosphorylated MLC protein of the Ang Ⅱ ＋ irbesartan and Ang Ⅱ ＋ Y27632 groups were （1.12 ±0. 09）and （1.22 ±0.10） respectively, both significantly lower than that of the Ang Ⅱ group （ 1.33 ± 0.06, both P 〈 0.01 ）. The level of phosphorylated MLC protein of the Ang Ⅱ ＋ ML-7 ＋ staur