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目的:采用电穿孔方法,介导Ras蛋白ssDNA适配子体外转染人大隐静脉平滑肌细胞,探讨外源性DNA转染血管平滑肌细胞的最适电转条件。方法:体外培养3-8代人大隐静脉平滑肌细胞,选择方波、1个脉冲、不同的电压及脉冲时间,应用电穿孔仪将Cy3标记的Ras蛋白ssDNA适配子转染至平滑肌细胞。采用CCK-8法观察细胞存活率,以及倒置荧光显微镜观察适配子转染效率。结果:细胞在电压160V、脉冲时间15ms时,细胞存活率最高,为(96.17±2.60)%;电压280V、脉冲时间25ms时,细胞存活率最低,为(30.04±2.58)%。细胞存活率随着电压的增大、脉冲时间的延长而降低。电压200V以下,转染率随电压增大、脉冲时间延长而升高,并于电压为200V、脉冲时间为25ms时,转染率到达峰值,为(84.41±5.16)%。但是,电压大于200V时,转染率却随电压增大、脉冲时间延长而降低,电压为280V、脉冲时间为25ms时,转染率降至最低,为(24.54±7.07)%。电压200V、脉冲时间20ms时,可获得最适转染效率及细胞存活率。结论:在方波,1个脉冲,适当的电压、脉冲时间下,电穿孔法转染血管平滑肌细胞可快速、简便地获得较高的转染效率及细胞存活率。电穿孔法是研究原代、化学法难转染平滑肌细胞生物学行为较为理想的转染方法。
OBJECTIVE: To investigate the optimal conditions of transfection of exogenous DNA into vascular smooth muscle cells by using electroporation method, and to mediate the transfection of ssDNA aptamers of Ras protein in human saphenous vein smooth muscle cells. Methods: 3-8 passages of human saphenous vein smooth muscle cells were cultured in vitro. The square wave, one pulse, different voltage and pulse time were selected. The Cy3-labeled ssDNA aptamer of Ras was transfected into smooth muscle cells by electroporation. The cell viability was observed by CCK-8 method and the transfection efficiency of aptamer was observed by inverted fluorescence microscope. Results: The cell survival rate was (96.17 ± 2.60)% when the voltage was 160V and the pulse time was 15ms. The cell survival rate was the lowest (30.04 ± 2.58)% when the voltage was 280V and the pulse time was 25ms. Cell survival rate decreases with increasing voltage and pulse time. Under the voltage of 200V, the transfection efficiency increased with the increase of voltage and pulse time, and reached 84.41 ± 5.16% at the voltage of 200V and the pulse time of 25ms. However, when the voltage was more than 200V, the transfection efficiency decreased with the increase of voltage and pulse time. The transfection rate was reduced to (24.54 ± 7.07)% when the voltage was 280V and the pulse time was 25ms. Voltage 200V, pulse time 20ms, the optimal transfection efficiency and cell survival can be obtained. CONCLUSION: Transfection of vascular smooth muscle cells by electroporation can achieve high transfection efficiency and cell survival rate rapidly and simply under the condition of square wave, 1 pulse, appropriate voltage and pulse time. Electroporation method is to study the primary, chemical method is difficult to transfect the smooth muscle cell biological behavior of the ideal method of transfection.