Ras蛋白适配子体外电转染人大隐静脉平滑肌细胞的初步研究

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160 V、脉冲时间15 ms时,细胞存活率最高,为(96.17±2.60)％；电压280 V、脉冲时间 25 ms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200 V以下,转染率随电压增大、脉冲时间延长而升高,并于电压为200 V、脉冲时间为25 ms时,转染率到达峰值,为(84.41±5.16)％.但是,电压大于200 V时,转染率却随电压增大、脉冲时间延长而降低,电压为280 V、脉冲时间为25 ms时,转染率降至最低,为(24.54±7.07)％.电压200V、脉冲时间20 ms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.

Electroporation Delivery of ssDNA Aptamers Targeting the Ras Protein into Human Saphenous Vein Smooth Muscle Cells in Vitro

Objective: To transfect ssDNA aptamers targeting Ras protein into human saphenous vein smooth muscle cells using by electroporation in vitro,and to study the optimal electroporation conditions for intracellular delivery of exogenous DNA in vascular smooth cells.Methods: Cy3 marked ssDNA aptamers targeting Ras protein was transfected into smooth muscle cells by electroporation under different electric voltages and pulse lengths.The cell survival rate and transfection efficiency were accessed by CCK-8 and inverted fluorescence microscope respectively.Results: Among all the conditions tested,160 V and 15 ms using a square wave form resulted in the highest cell survival rate as(96.17±2.60) %,while 280 V and 25 ms resulted in the lowest cell survival rate as(30.04±2.58) %.With the increasing of voltages and the prolonging of pulse lengths,the cell survival rate decreased gradually.Under conditions below 200 V,transfection efficiency increased when voltages increased and pulse lengths prolonged.Nevertheless,when above 200 V,it decreased when voltages increased and pulse lengths prolonged.When voltage was 200 V and pulse length was 25 ms,transfection efficiency reached the peak as(84.41±5.16) %,while 280 V and 25 ms,it aome to the lowest value as(24.54±7.07) %.From all the datum,the optimal electroporation conditions were 200 V and 20 ms using a square wave form and one pulse.Conclusion: Under optimal electric voltage and pulse length,we could access ideal transfection efficiency and cell survival rate efficiently and simply.Electroporation is an efficient transfection method for primary and other difficult-to-transfect cells.