过表达LncRNA GAS5对SD大鼠心肌成纤维细胞活化增殖的影响

目的 应用长链非编码RNA GAS5 (LncRNA GAS5)过表达质粒(pEGFP-C1-GAS5)转染SD大鼠心肌成纤维细胞,观察其对心肌成纤维细胞活化增殖的影响.方法 提取SD大鼠心肌成纤维细胞,转化生长因子β1 (TGF-β1)刺激其增殖,分为pEGFP-C1-GAS5组、阴性对照组和空白对照组,应用LncRNA GAS5基因的过表达质粒 (pEGFP-C1-GAS5)通过脂质体瞬时转染细胞,48 h后收获细胞.采用实时定量聚合酶链反应(qRT-PCR)检测GAS5、Ⅰ型胶原前胶原(Col1A1)和α-平滑肌肌动蛋白(α-SMA)的mRNA表达,Western blot分析Col1A1和α-SMA蛋白表达变化,MTT法检测细胞增殖活力.结果 TGF-β1刺激心肌成纤维细胞后,qRT-PCR结果显示 LncRNA GAS5的表达量较正常组下降(P<0.05);与阴性对照组和空白对照组比较,转染了pEGFP-C1-GAS5的实验组,qRT-PCR结果显示实验组的GAS5表达均明显升高(P<0.05),同时α-SMA和Col1A1 mRNA表达显著降低(P<0.05);Western blot结果显示转染pEGFP-C1-GAS5的实验组α-SMA和Col1A1蛋白表达量均明显降低(P<0.05);MTT法检测结果表明在实验组心肌成纤维细胞的增殖活力低于阴性对照组和空白对照组(P<0.05).结论 过表达LncRNA GAS5可显著降低心肌成纤维细胞的增殖活力,提示GAS5有抑制心肌成纤维细胞的活化增殖的作用,为以后进一步研究提供依据.

Effect of overexpression of LncRNA GAS5 on proliferation andactivation of cardiac fibroblasts in SD rats

Objective To transfect the cardiac fibroblasts (CFs) in vitro with the overexpression plasmid pEGFP-C1-GAS5, and investigate its effect on activation and proliferation of cardiac fibroblasts in rats.Methods Cardiac fibroblasts were extracted from SD neonate rats and cultured.Transforming growth factor-β1 (TGF-β1) was used to stimulate their proliferation.CFs were divided into pEGFP-C1-GAS5 group, negative control group and blank control group.Lipofectamine 2000 Reagent was used to transfected CFs with overexpression plasmid pEGFP-C1-GAS5, and cells were harvested after 48 hours.The expressions of GAS5 and mRNA of α-SMA and Col1A1 were analyzed by qRT-PCR.The expressions of α-SMA and Col1A1 in protein level were detected by Western blot.The activities of cells proliferation were confirmed by MTT assay.Results After stimulate cardiac fibroblasts with TGF-β1, qRT-PCR showed that the expression of LncRNA GAS5 than the normal CFs decreased.Compared with the negative and no-treatment control group, qRT-PCR results showed that the expression of LncRNA GAS5 was significantly increased in pEGFP-C1-GAS5 transfected CFs(P<0.05), and down-regulated the mRNA levels of α-SMA and Col1A1(P<0.05).Results from Western blot demonstrated significant decrease of α-SMA and Col1A1 in the group that cells transfected with pEGFP-C1-GAS5 (P<0.05).MTT assay results suggested that in the experimental group the CFs proliferation activity decreased significantly than the negative and no-treatment control group (P<0.05).Conclusion Overexpression of LncRNA GAS5 can significantly weaken the proliferation activity of cardiac fibroblasts, which indicates that GAS5 can inhibit CFs proliferation activity, and provide the basis for further studies.