人永生化表皮细胞HaCaT电转染条件的优化

Optimization of electrotransfection conditions for the immortal human keratinization HaCaT cell line

目的比较不同电转染条件下,人永生化表皮细胞HaCaT的转染效率,探讨影响HaCaT电转染率和存活率的主要因素,筛选出HaCaT细胞最佳的电转染条件。方法应用电穿孔仪,设定不同的电压(180、360、540、720V)、脉冲时间(40、60、80、100、120μs)、脉冲周期(1、2、3、4、5次)和温度(室温和4℃)等条件,将质粒(pGPU6/GFP/nev)电转染至HaCaT细胞。电转染24h后,荧光镜下和光镜下分别计算绿色荧光阳性细胞数和总细胞数,并随机取3个视野算平均值,计算电转染率;MTT法检测电转染后细胞的存活率。结果当HaCaT细胞浓度为1.5×106/mL,pGPU6/GFP/nev质粒浓度为2.0μg/mL,HaCaT细胞的最佳电转染条件为540V/60μs/Pulse1/RT,此时细胞生长状态最佳,电转染率可达到33.23%,细胞存活率为61.22%。结论本实验优化了HaCaT细胞电转染的最佳条件,提高了HaCaT细胞电转染效率,为后续基因功能的研究奠定了基础。

Objective To investigate the method and optimum conditions of electrotransfection and the major influential factors of electroransfection efficiency and the survival rate of human immortalized epidermal keratinization cells.Methods The pGPU6/GFP/nev plasmids were electrotransfected into HaCaT by electroporation apparatus with different electric voltages （180,360,540 and 720 V）,time of pulse （40,60,80,100 and 120 μs）,electronic shock times （1,2,3,4 and 5）,and incubation temperature （room temperature and 4 ℃）.The number of green fluorescence positive cells and the total cell number were counted respectively under fluorescent microscope and visible light microscope after 24h,the electrotransfection efficiency was counted by the average of three random fields of vision; cell viability was measured by MTT assay.Results 540 V/60 μs/Pulse1/RT were found to be the optimal electric transfection conditions in HaCaT cell when the concentration of HaCaT cell and pGPU6/GFP/nev was 1.0 × 106 cells/ml and 2.0 μg/mL.HaCaT cell had a better cellular growth behavior,and electrotransfection rate and cell survival rate were about 33.23％ and 61.22％,respectively.Conclusion By optimizing the electransfection conditions,the electransfection rate of HaCaT cell is efficiently improved,which will lay the foundation for further studying gene functions.