电穿孔法转染脐带华通胶间充质干细胞的最佳条件  

Optimal conditions for transfecting Wharton's Jelly-derived mesenchymal stem cells with electroporation method

作　　者：王泽 李春花 张宁坤 高连如 陈宇[1,2] Wang Ze;Li Chun-hua;Zhang Ning-kun;Gao Lian-ru;Chen Yu(The Navy Clinical College of Anhui Medical University,Beijing 100048,China;Department of Cardiology,Navy General Hospital of PLA,Beijing 100048,China)

机构地区：[1]安徽医科大学海军临床学院,北京市100048 [2]解放军海军总医院心血管内科,北京市100048

出　　处：《中国组织工程研究》2018年第17期2717-2721,共5页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金(81370238;81370901)~~

摘　　要：背景:脐带华通胶间充质干细胞是目前较为原始的干细胞,为当前干细胞的研究热点之一,是基因治疗的理想载体。然而,目前尚未有对脐带华通胶间充质干细胞电转染条件的研究,因此探索脐带华通胶间充质干细胞的电转染最佳条件占据重要地位。目的:探讨不同电穿孔条件对脐带华通胶间充质干细胞转染率的影响,以确定最佳电转染条件。方法:通过控制电压、脉冲时长及细胞状态等转染条件,采用不同条件组合后用电穿孔法将质粒EEV-EGFP转入脐带华通胶间充质干细胞,流式细胞仪检测转染率。结果与结论:(1)电压自125 V至150 V,转染率呈现递增趋势,150 V时获得最大转染率,但将电压进一步调高至170 V,转染率开始显著降低;(2)脉冲时长为5.0 ms时获得最大转染率;脉冲时长为2.5 ms和7.5 ms时转染效率均有所降低;(3)与低氧培养比较,正常培养的细胞转染率更高;(4)结果表明,正常培养的脐带华通胶间充质干细胞在电压150 V、脉冲时长5.0 ms、脉冲间隔时间50 ms、脉冲数2次时可达到较高的电转染率。BACKGROUND： Wharton＇s Jelly-derived mesenchymal stem cells are relatively primitive stem cells that are ideal vectors for gene therapy. However, there is a lack of studies on the conditions for the electrotransfection of Wharton＇s Jelly-derived mesenchymal stem cells. Therefore, exploring the optimal conditions for the electrotransfection of Wharton＇s Jelly-derived mesenchymal stem cells occupies an important position. OBJECTIVE： To investigate the effects of different electroporation conditions on the transfection efficiency of Wharton＇s Jelly-derived mesenchymal stem cells, and to explore the optimal conditions for cell electroporation. METHODS： By controlling the transfection conditions such as voltage, pulse duration and cell status, EEV-EGFP plasmids were transfected into Wharton＇s Jelly-derived mesenchymal stem cells by electroporation under different conditions. Transfection efficiency was detected by flow cytometry. RESULTS AND CONCLUSION：（1） The transfection efficiency was intended to increase when the voltage ranged from 125 V to 150 V, and the maximum transfection efficiency was obtained when the voltage was 150 V. However, when the voltage was further increased to 170 V, the transfection efficiency began to decrease considerably.（2） The maximum transfection efficiency was obtained when the pulse duration was 5.0 ms, while it was certainly decreased when the pulse duration was 2.5 and 7.5 ms.（3） The transfection efficiency of the cells cultured under normoxia was higher than that under hypoxic culture. These findings reveal that normally cultured Wharton＇s Jelly-derived mesenchymal stem cells can achieve higher electroporation efficiency via two pulse sessions at a voltage of 150 V, pulse duration of 5.0 ms, and pulse interval time of 50 ms.

关 键 词：脐带 间质干细胞 转染 组织工程 电穿孔 电转染 华通胶间充质干细胞 华通胶 低氧培养 干细胞 国家自然科学基金 

分 类 号：R394.2[医药卫生—医学遗传学]