脂联素随原代HSC活化表达的动态变化及其对原代HSC表达MMP-13的影响

Expression of adiponectin during hepatic stellate cell(HSC)activation and the effect of adiponectin on expression of matrix metalloproteinase-13 in primary HSCs

目的:探讨脂联素mRNA在原代肝星状细胞(hepatic stellate cell,HSC)活化过程中表达的变化趋势,以及其对体外培养的原代HSC增殖状况和金属基质蛋白酶-13(MMP-13)mRNA与蛋白表达的影响.方法:改良的肝脏二步原位灌注法分离培养原代HSC;以α-SMA为原代HSC活化的标志,Real-time PCR方法检测随HSC活化脂联素表达的动态变化;MTT方法检测脂联素对活化HSC增殖的影响,以外源性脂联素处理原代HSC,根据脂联素浓度,分为对照组,脂联素处理浓度62.5μg/L,125μg/L,250μg/L,500μg/L五组,Real-time PCR方法检测MMP-13 mRNA表达;ELISA方法检测原代HSC分泌的MMP-13蛋白量.结果:HSC存活率的下降与脂联素浓度呈线性相关(OR=0.828).MMP-13 mRNA和蛋白表达水平与外源性加入脂联素浓度正相关,当脂联素浓度增加至500 g/L时,MMP-13 mRNA表达增高至对照组的5.54倍,MMP-13含量增加至30.951μg/L显著高于对照组的24.127μg/L,差异具有显著统计学意义.结论:脂联素具有抑制肝纤维化的作用,其机制可能与抑制HSC增殖和增强MMP-13在HSC细胞的表达有关.

AIM： To study trends in the expression of adiponectin during hepatic stellate cell （HSC） activation and the effects of adiponectin on mRNA and protein expression of matrix metalloproteinase-13 （MMP-13）, which was closely related to liver fibrosis in primary HSCs. METHODS： We isolated primary HSCs through improved two-step liver in situ perfusion, and investigated changes in expression of adiponectin during activation of primary HSCs. MTT was used to study the effect of adiponectin on proliferation of activated HSCs. We chose proper concentration of adiponectin and then divided primary HSCs into five groups according to the concentration of adiponectin. Real-time PCR was used to detect the difference of α-smooth muscle actin, adiponectin and MMP-13 expression between the groups. Enzyme linked immunosorbent assay （ELISA） was used to detect the quantity of MMP-13 pro-tein secreted into the medium. RESULTS： The survival rate of HSCs showed a linear correlation with adiponectin concentration （OR = 0.828）. Expression of adiponectin was down-regulated during HSC activation. Adiponectin was able to inhibit the proliferation of HSCs. When the concentration of adiponectin was 500 μg/L, MMP-13 mRNA expression, in contrast with that in the control group, increased by 5.54 times. MMP-13 protein expression in adiponectin-treated cells increased significantly in contrast with that in the control group （30.951 μg/L vs 24.127 μg/L）. CONCLUSION： Expression of adiponectin was decreased during HSC activation. As a member of the adipokines, adiponectin can exert its potential anti-fibrotic activity through inhibition of HSC proliferation and up-regulation of expression of MMP-13 in primary HSCs.