胆固醇结石病小鼠胆囊组织中Rho-ROCK信号、钙调信号相关分子的表达及意义

Expression and significance of Rho-ROCK signal and calmodulin-related molecules in gallbladder tissues of mice with cholesterol gallstone

目的观察胆固醇结石病(胆石病)小鼠胆囊组织中Rho-ROCK信号和钙调信号相关分子的表达,探讨其在胆固醇结石形成过程中的作用。方法将40只C57BL/6小鼠随机分为对照组和胆石组各20只,对照组给予正常饮食,胆石组给予高脂饮食诱导胆石病模型,连续8周。小鼠麻醉,剖腹观察胆囊的结石形成情况。胆总管插管,收集胆汁,采用荧光探针检测胆汁中的钙离子(Ca^2+)水平;眼球取血,采用HITACHI自动分析仪检测胆汁和血清总胆固醇和高密度胆固醇(HDL-C)含量,并计算非高密度脂蛋白胆固醇含量。小鼠脱颈椎处死,取胆囊组织,采用HE染色法观察胆囊组织的病理变化;采用RT-PCR法和Western blotting法检测胆囊组织中Rho-ROCK信号相关分子(RhoA、RhoB、ROCK)和钙调信号相关分子[肌球蛋白轻链激酶(MLCK)、磷酸化的肌球蛋白轻链(p-MLC)、肌球蛋白轻链磷酸酶(MLCP)]mRNA及蛋白表达。结果对照组胆囊无结石和胆泥形成,胆石组胆囊出现胆结石6只、胆泥5只,其余9只未造模成功被排除。与对照组相比,胆石组胆汁分泌率和胆汁中的Ca 2+水平下降,胆汁和血清总胆固醇、非高密度脂蛋白胆固醇升高(P均<0.05)。病理检查显示,胆石组胆囊组织出现炎症细胞浸润及胆囊壁增厚等炎症迹象。胆石组胆囊组织中RhoA、RhoB、ROCK、MLCK、p-MLC mRNA及蛋白表达较对照组下降,MLCP mRNA及蛋白表达较对照组升高(P均<0.05)。结论胆石病小鼠胆囊组织中Rho-ROCK信号和钙调信号相关分子的表达发生异常变化,Rho-ROCK和钙调信号相关分子可能参与了胆固醇结石的形成过程。

Objective To observe the expression of Rho-ROCK signal and calmodulin-related molecules in gallbladder tissues of mice with cholesterol gallstone,and to explore its role in the formation of cholesterol stones.Methods C57BL/6 mice were randomly divided into the control group and gallstone group.The mice in the control group were given normal diet and the gallstone group with high fat diet for 8 weeks.The mice were anesthetized,the formation of gallstones in the gallbladder was observed by laparotomy,the bile duct was inserted,the bile was collected,the blood was taken from the eyeball,the cervical vertebrae were sacrificed,the gallbladder tissue was separated,and the incidence of stones was calculated.Fluorescence probe was used to detect calciumion(Ca^2+)levels in bile;HITACHI automatic analyzer was used to detect total cholesterol and high-density cholesterol(HDL-C)in the bile and serum,and the content of non-high-density cholesterol was calculated.The pathological changes of gallbladder tissues were detected by HE staining.The mRNA and protein expression levels of Rho-ROCK and calmodulin-related molecules in gallbladder tissues were detected by RT-PCR and Western blotting,respectively.Results There were no stones and biliary sludge in the gallbladder of the control group.In the gallbladder of the gallstone group,6 mice were found to have gallstones,5 had only biliary mud,and the remaining 9 models were unsuccessful and were excluded.Compared with the control group,the incidence of gallbladder mud or gallstone formation and gallstone in gallstone group increased significantly,but the bile secretion rate and Ca^2+levels in bile decreased significantly,the bile and serum total cholesterol,non-high density of cholesterol increased in the gallbladder group(all P<0.05).Pathological examination showed inflammatory cell infiltration and gallbladder wall thickening and other inflammatory signs were found in the gallstone tissues of the gallstone group.The mRNA and protein expression levels of RhoA,RhoB,ROCK,myosin light chain kinase(MLCK)and phosphorylated myosin light chain(p-MLC)were down-regulated,and myosin light chain phosphatase(MLCP)mRNA and protein expression levels were up-regulated in the gallbladder tissue of the gallstone group(all P<0.05).Conclusion The expression of Rho-ROCK signal and calmodulin-related molecules in gallbladder tissues of mice with cholesterol gallstone is abnormal,and Rho-ROCK and calcium signaling related molecules may be involved in the formation of cholesterol gallstone.