脂肪特异性蛋白27对大鼠肝星状细胞活化的影响

Effect of fat-specific protein 27 on activation of rat hepatic stellate cells in vitro

目的:探讨脂肪特异性蛋白27(Fsp27)对肝星状细胞(HSCs)增殖和活化的影响及其对纤维化相关蛋白的调节作用。方法:从SD大鼠肝脏提取HSCs并培养,采用实时荧光定量PCR、免疫荧光染色和Western blotting检测原代HSCs和活化HSCs中Fsp27 mRNA和蛋白的表达。构建携带Fsp27基因的慢病毒,转染活化的HSCs并继续培养72 h,通过CCK-8比色法检测Fsp27对HSCs增殖的影响;Western blotting检测HSCs中α-平滑肌肌动蛋白(α-SMA)的表达,了解HSCs的活化状态;实时荧光定量PCR检测Fsp27对HSCs中纤维化相关蛋白[包括基质金属蛋白酶2(MMP-2)、金属蛋白酶组织抑制物1(TIMP-1)和转化生长因子β1(TGF-β1)]mRNA表达的影响。结果:成功分离大鼠原代HSCs。Fsp27在原代HSCs和活化HSCs中的表达差异显著(P<0.01);活化HSCs成功转染携带Fsp27基因的慢病毒后继续培养72 h,与对照组比较,HSCs的活化与增殖被明显抑制(P<0.05);Fsp27促进MMP-2 mRNA的表达(P<0.05),降低TIMP-1和TGF-β1mRNA的表达(P<0.05)。结论:Fsp27可抑制HSCs的增殖和活化,并调节纤维化相关蛋白的表达。Fsp27的作用可能与其维持HSCs静息状态细胞表型有关。

AIM：To investigate the effects of fat-specific protein 27 （Fsp27） on the proliferation and activation of hepatic stellate cells （HSCs） in vitro. METHODS：HSCs were isolated from the liver of SD rats. The mRNA and protein expression of Fsp27 in primary HSCs and activated HSCs was detected by real-time fluorescence quantitative PCR, immunofluorescence staining and Western blotting. After 72 h of transfection with Fsp27-carrying lentivirus （pLV-Fsp27）, the proliferation of HSCs was tested by CCK-8 assay, the protein expression of α-smooth muscle actin （α-SMA） in HSCs was detected by Western blotting, and the mRNA expression of fibrosis-related proteins, including matrix metalloproteinase 2 （MMP-2）, tissue inhibitor of metalloproteinase 1 （TIMP-1） and transforming growth factor beta 1 （TGF-β1）, was determined by real-time fluorescence quantitative PCR. RESULTS：Rat HSCs were successfully isolated and cultured. The difference of Fsp27 expression between primary HSCs and activated HSCs was significant （P〈0.01）. The proliferation and activation of HSCs was inhibited 72 h after pLV-Fsp27 transfection （P〈0.05）. Fsp27 enhanced the mRNA expression of MMP-2 and down-regulate the mRNA expression of TIMP-1 and TGF-β1 in activated HSCs （P〈0.05）. CONCLUSION：Fsp27 inhibits the proliferation and activation of HSCs and regulates the expression of fibrosis-related proteins. Fsp27 may play an important role in maintenance of the quiescent phenotype of HSCs.