山东医药2016，Vol.56Issue(47)：13-16,4.DOI:10.3969/j.issn.1002-266X.2016.47.004
食管癌Eca109细胞的HPV16-E6基因转染条件优化
Optimal transfection conditions for HPV1 6-E6 gene in esophageal cancer Eca1 09 cells
摘要
Abstract
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.关键词
食管癌/人乳头瘤病毒16/基因转染效率/细胞相对存活率/实验设计Key words
esophageal cancer/human papillomavinus 16/gene transfection efficiency/cell relative survival rate/experimental design分类
医药卫生引用本文复制引用
康馨丹,陈卫刚,阮开学,张宁,郑勇..食管癌Eca109细胞的HPV16-E6基因转染条件优化[J].山东医药,2016,56(47):13-16,4.基金项目
国家自然科学基金资助项目（81260362）。 （）
