视黄醇类核内受体-α基因慢病毒载体的构建及其对肝星状细胞的影响

Construction of retinoid X receptor-alpha lentivirus vector and its effects on hepatic stellate cells

目的:通过视黄醇类核内受体-α(retinoid X receptor-alpha,RXR-α)基因慢病毒表达载体的构建,及其对肝星状细胞(hepatic stellate cells,HSC)活化、增殖影响的研究,探讨RXR-α基因在肝纤维化中的作用。方法:①构建RXR-α基因慢病毒表达载体;②分离和体外培养大鼠HSC;③将自发活化的HSC分成3组,即正常对照组、阴性对照组和RXR-α载体组;分别将空白慢病毒载体和RXR-α慢病毒载体转染自体活化的HSC,采用Western印迹法检测各组细胞中RXR-α、α-SMA和Ⅰ型胶原蛋白表达变化,用MTT法检测各组HSC培养24、48、72及96 h后的增殖情况。结果:正常对照组及阴性对照组HSC中几乎没有RXR-α蛋白的表达,而RXR-α载体转染组的HSC中出现RXR-α蛋白的明显表达。与正常对照组及阴性对照组相比,RXR-α载体组转染的HSC中α-SMA蛋白表达量显著下降(t=3.767,P<0.01和t=8.491,P<0.05),Ⅰ型胶原蛋白表达也显著降低(t分别为10.449和10.756,P值均<0.01),同时HSC的增殖能力也显著下降(t分别为4.381和1.778,P值均<0.05)。而阴性对照组与正常对照组相比,α-SMA和Ⅰ型胶原蛋白表达量和HSC增殖能力无明显变化(P>0.05)。结论:RXR-α基因在HSC内的增强表达能显著抑制HSC的活化和增殖,具有潜在的抑制肝纤维化的作用。

Objective To evaluate the role of RXR-α in hepatic fibrogenesis,through construction of RXR-α expression Lentivirus vector and research for the effects of RXR-α on HSC.Methods ① Construction of the RXR-α expression Lentivirus vector.② The rat HSC were isolated and cultured in vitro,and the HSC through autoactivation of several days for the next experiment.③ HSC were divided into three groups： normal controls（CON）,negative controls（NC） and the LV-RXR-α（RXR-α） group.Non-RXR-α emply lentivirus vector and RXR-α lentivirus vector were transfected into HSC of autoactivation separately.The protein expression of RXR-α,α-SMA and collagen-Ⅰ were detected by Western blotting.Meanwhile,after cultivation of 24 h,48 h,72 h and 96 h HSC proliferation was analyzed by MTT assay.Results After transfection of RXR-α lentivirus vector,RXR-α protein expresed in HSC was upregulated obviously.Compared with the normal control and the negative control groups,the protein expression of α-SMA was decreased significantly（t=3.767,P〈0.01 and t=8.491,P〈0.05）,and the expression of collagen-Ⅰwas also reduced significantly（t=10.449 and t=10.756,both P〈0.01）.Meanwhile,the proliferation of HSC was also markedly suppressed（t=4.381 and t=1.778,both P〈0.05）.However,compared with the normal control groups,none of them was markedly suppressed in the negative control groups（P〉0.05）.Conclusions The expression of RXR-α gene can significantly suppress the activation and proliferation of HSC,and potentially attenuate hepatic fibrogenesis.Thus,promoting the expression of RXR-α in vivo could be a new approach to treat hepatic fibrosis.