Objective:To investigate the role of overexpression of Notch3 in the transformation of cardiac fibroblasts (CFs) to myofibroblasts (MFs).Methods:CFs were isolated by collagenase digestion method and incubated for 24 h with 10 ng/ml TGF-β1.Hydroxyproline kit was used to measure hydroxyproline content in the supernatants,immunofluorescence was used to measure α-smooth muscle actin (αSMA) expression,western blotting analysis was used to measure Notch3 expression.Lentiviral was used to transfect CFs,overexpression of Notch3.When the multiplicity of infection (MOI) was 20,transfected into 72 h was the optimal transfection condition,fluorescence microscopy was used to observe the expression of green fluorescent protein (GFP),western blotting analysis was used to measure the efficiency of Notch3 overexpression.Hydroxyproline kit was used to measure hydroxyproline content in the supernatants,immunofluorescence was used to measure α-SMA expression.Results:The 10 ng/ml TGF-β1 induced increases of hydroxyproline content in the supernatants and α-SMA expression significantly in CFs and induced decreases of Notch3 expression.Compared with the blank control group,there were significant differences (P＜0.05).Lentiviral transfection of Notch3 overexpression,compared with the blank control group,hydroxyproline content in the supernatants and α-SMA expression decreased in overexpression group (P ＜ 0.05).Conclusion:overexpression of Notch3 can inhibit the transformation of cardiac fibroblasts to myofibroblasts.%目的:探讨Notch3过表达对心肌成纤维细胞(CFs)向肌成纤维细胞(MFs)转化的作用.方法:用胶原酶消化法分离培养SD仔鼠的CFs,将CFs于10 ng/ml转化生长因子-β1(TGF-β1)中孵育24 h,用羟脯氨酸试剂盒检测细胞上清羟脯氨酸含量,免疫荧光法检测CFs中平滑肌肌动蛋白(α-SMA)表达,用Western-blotting检测Notch3蛋白表达;利用慢病毒转染CFs,使其过表达Notch3,确定最佳转染条件为感染复数(MOI)20,转染时间72 h,荧光显微镜观察绿色荧光蛋白(GFP)表达,Western-blotting检测Notch3过表达效率;用羟脯氨酸试剂盒检测细胞上清羟脯氨酸含量,免疫荧光法检测CFs中α-SMA表达.结果:10 ng/ml TGF-β1可显著诱导CFs细胞上清羟脯氨酸含量及α-SMA表达增加,同时使Notch3表达下降,与空白对照组相比有统计学差异(P＜0.05).慢病毒转染CFs过表达Notch3后,与空白对照组相比,Notch3过表达组细胞上清羟脯氨酸含量及α-SMA表达均显著减少(P＜0.05).结论:过表达Notch3能抑制CFs向MFs转化.
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