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目的:体外HCN2基因转染人脂肪干细胞(Adipose-derived stem cells,ADSCs)构建起搏细胞。方法:取人脂肪组织分离得到脂肪干细胞并进行体外培养,Ad.HCN2转染后检测HCN2表达情况,检测转染后ADSCs自主产生起搏电流(pacemaker current,If)的能力。结果:RT-PCR结果显示实验组HCN2基因表达。Western-blot可检测到实验组HCN2蛋白表达。流式细胞仪检测可见实验组HCN2阳性比例明显升高。免疫荧光检测可见转染的ADSCs发出红色荧光。膜片钳可记录到起搏电流,其激活电位约为-60 mV,完全激活电位140mV,呈电压依赖性。结论:ADSCs被Ad.HCN2转染后可表达目的基因HCN2,并自主产生起搏电流。
OBJECTIVE: To construct pacemaker cells by transfecting human adipose-derived stem cells (ADSCs) with HCN2 gene in vitro. Methods: Adipose-derived stem cells were isolated and cultured in vitro. The expression of HCN2 was detected by Ad.HCN2 transfection, and the pacemaker current (If) was independently produced after ADSCs transfection. Results: RT-PCR results showed HCN2 gene expression in experimental group. Western-blot detected HCN2 protein expression in the experimental group. Flow cytometry showed that the experimental group HCN2 positive ratio was significantly higher. Immunofluorescence showed that the transfected ADSCs emitted red fluorescence. Patch clamps record pacing currents with an activation potential of approximately -60 mV and a fully activated potential of 140 mV in a voltage-dependent manner. CONCLUSION: ADSCs transfected with Ad.HCN2 can express HCN2 and produce pacing current autonomously.