慢病毒转导敲低Smad7表达对大鼠原代心肌成纤维细胞增殖、迁移、细胞表型分化和Ⅰ型胶原蛋白表达的影响  

Effect of Smad7 deficiency on rat cardiac fibroblasts proliferation,migration,cell differentiation and collagenⅠsecretion in vitro

作　　者：罗红[1,2] 高歌 张光琼 刘欢 杨红宇[2] 沈祥春 LUO Hong;GAO Ge;ZHANG Guang-qiong;LIU Huan;YANG Hong-yu;SHENG Xiang-chun(High Educational Key Laboratory of Guizhou Province for Natural Medicinal Pharmacology and Druggability,Guizhou Medical University,Guiyang 550025,China;Laboratory Animal Center of Guizhou Medical University,Guiyang 55004,China)

机构地区：[1]贵州医科大学贵州省高等学校天然药物药理与成药性评价特色重点实验室,贵阳550025 [2]贵州医科大学实验动物中心,贵阳550025

出　　处：《解剖学报》2022年第5期578-584,共7页Acta Anatomica Sinica

基　　金：贵阳市科技计划项目(筑科合同[2017]30-17);贵州省科技合作计划项目(黔科合LH字[2016]7356);中央引导地方科技科技发展专项基金(黔科中引地[2016]4007)。

摘　　要：目的探讨Smad7敲低后对原代心肌成纤维细胞增殖、迁移、胶原分泌和细胞表型转化的影响。方法原代培养10只SD大鼠乳鼠的心肌成纤维细胞,免疫组织化学染色鉴定细胞。采用慢病毒转染方法敲低心肌成纤维细胞Smad7的表达,Western blotting验证Smad7蛋白敲低效率,实时无标记细胞仪检测心肌成纤维细胞的增殖情况,细胞划痕实验检测细胞的迁移情况,Western blotting检测Ⅰ型胶原蛋白(ColⅠ)和细胞表型转化标志物α-平滑肌肌动蛋白(α-SMA)的表达情况。结果成功培养心肌成纤维细胞,并进行细胞鉴定。慢病毒转染的心肌成纤维细胞感染复数(MOI)值为100,转染后88.33%的细胞表达绿色荧光蛋白,慢病毒敲低组Smad7的蛋白表达明显下调(P<0.05)。慢病毒敲低Smad7后细胞增殖明显下降(P<0.01),细胞迁移率明显减少(P<0.01)。与对照组相比,慢病毒敲低组细胞ColⅠ表达明显下降(P<0.01),α-SMA表达明显升高(P<0.01)。结论Smad7表达敲低后,心肌成纤维细胞的细胞功能发生明显改变,这些改变可能与Smad7下调导致心肌纤维化程度加重有关。Objective To investigate the effects of Smad7 knock down by lentivirus on rat cardiac fibroblasts proliferation,migration,cell differentiation and collagen secretion in vitro.Methods The primary cardiac fibroblasts were separated from the hearts of ten SD rats and identified by immunohistochemical method.The lentivirus transfection knocked down the expresson of Smad7 in cardiac fibroblasts,Western blotting was used to detect the efficiency of Smad7 knock down by lentivirus.The proliferation of cardiac fibroblasts was quantified by real-time unlabeled cell analyzer.Cell migration was evaluted by cell wound scratch assay.Western blotting was used to detect expression ofα-smooth muscle actin(α-SMA)and collagenⅠ(ColⅠ).Results Myocardial fibroblasts were successfully cultured and identified by immunocytochemical methods.The multiplicity of infection(MOI)that lentivirus transduction of myocardial fibroblasts was 100.After lentivirus transduction,88.33%myocardial fibroblasts expressed green fluorescent protein,showed that the lentivirus could significantly reduce the protein expression of Smad7.Smad7 deficiency decreased the proliferation and migration of cardiac fibroblasts,increased the protein expression ofα-SMA and decreased collagen secretion.The results indicated that Smad7 deficiency significantly down-regulated the proliferation and migration of cardiac fibroblasts,increasedα-SMA protein expression and reduced ColⅠprotein expression.Conclusion Smad7 deficiency can significantly change the cardiac fibroblasts function,that is related to the pathological mechanism that lead to myocardial fibrosis.

关 键 词：心肌成纤维细胞 慢病毒沉默 SMAD7 α-平滑肌肌动蛋白 Ⅰ型胶原蛋白 实时无标记细胞分析术 细胞划痕实验 大鼠 

分 类 号：Q813[生物学—生物工程]