食管癌Eca109细胞的HPV16-E6基因转染条件优化

摘要

目的：确定HPV16-E6基因转染食管癌Eca109细胞的最佳转染条件。方法取体外培养的食管癌Eca109细胞，用中心复合试验设计分组，转染条件控制因素包括DNA用量、Lip2000／DNA和饥饿时间，DNA用量分0．65、1．00、1．50、2．00、2．35μg共5档，Lip2000／DNA分0．65、1．00、1．50、2．00、2．35μL／μg共5档，饥饿时间分0、3、7．5、12、15 h共5档，共设20个组。转染24 h后，倒置显微镜下观察各组荧光情况并拍照，用图像处理软件计算细胞转染效率；用CCK-8法测算各组细胞相对存活率。用响应曲面法绘制有交互影响的因素对细胞转染效率和相对存活率影响的响应曲面，以目的基因细胞数（转染效率×细胞存活率）最大化为原则，确定最佳转染条件。在最佳转染条件下用HPV16-E6基因转染食管癌Eca109细胞，检测细胞转染效率和相对存活率。结果随着饥饿时间延长，细胞转染效率和细胞相对存活率呈下降趋势。依据饥饿0h的转染效率、细胞存活率响应曲面图，得出最佳转染条件为（24孔培养板）：DNA用量1．35μg、Lip2000／DNA1．12μL／μg（换算成Lip2000用量为1．51μL）、细胞饥饿时间0 h；转染效率预测值可达38．68％，细胞相对存活率预测值为85．09％。在最佳转染条件下用相同方法转染，实际转染效率为39．79％，与预测值的相对误差为2．87％。结论 HPV16-E6基因转染Eca109细胞的最佳转染条件（24孔板）为：每孔DNA用量1．35μg，脂质体Lip2000用量1．51μL，不对细胞进行饥饿处理。%Objective To find the optimal transfection conditions for transfecting HPV16-E6 plasmid to esophageal cancer Eca109 cells.Methods The in vitro cultured Eca109 cells were selected.We used central composite experiment to design the groups.The control factors of transfection conditions included DNA dosage (0.65,1.00,1.50,2.00,and 2.35 μg),Lip2000/DNA (0.65,1.00,1.50,2.00,and 2.35 μL/μg)and starvation time (0,3,7.5,12,and 15 h) and according to the above,we established 20 groups.After 24 h,the expression of green fluorescent was observed by in-verted microscope and then was photographed.The transfection efficiency was evaluated by image processing software,and the cell proliferation by CCK-8.Response surface methodology was used to draw the response surface on effects of interac-tion factors on cell transfection efficiency and relative survival rate.The optimal transfection conditions were determined based on the principle of maximizing the number of target cells (transfection efficiency ×cell survival rate).The transfec-tion efficiency and relative survival rate of esophageal carcinoma Eca109 cells were detected by HPV16-E6 gene transfection under optimal transfection conditions.Results On the basis of cell transfection efficiency without starvation,the response surface showed that the highest transfection efficiency area was located in the middle of the response surface.Without star-vation,the response surface of cell survival rate showed that the DNA and Lipo 2000 was negatively correlated with the sur-vival rate.The optimal transfection conditions (24-well plate)determined based on the principle of maximizing the number of target cells (transfection efficiency ×cell survival rate):the plasmid amount was 1.35 μg,Lipo2000/DNA ratio was 1.12∶1 μg/μL (converted to Lip2000 dosage of 1.51 μL)and the starvation time was 0 h.The predicted value of trans-fection efficiency reached 38.68% and the cell survival rate was 85.09%.Under the optimal transfection conditions,the actual transfection efficiency was the highest (39.79%),with the relative error of 2.87% as compared with the predicted value.Conclusion The optimal transfection conditions for transfecting HPV16-E6 plasmid to Eca109 cells for 24-well plate are asfollows:1 .35 μg DNA per well,1 .5 1 μL Lip2000 liposome,and the cells are not starved.