腺病毒介导的人表皮生长因子基因转染对人表皮细胞生物学特性的影响  

Effects of transfection of human epidermal growth factor gene with adenovirus vector on biological characteristics of human epidermal cells

作　　者：尹凯[1] 马丽[1] 申传安[1] 尚玉茹[1] 李大伟[1] 李龙珠[1] 赵东旭[1] 程文凤[1] 

机构地区：[1]解放军总医院第一附属医院全军烧伤研究所,北京100048

出　　处：《中华烧伤杂志》2016年第5期305-311,共7页Chinese Journal of Burns

基　　金：国家自然科学基金面上项目（30971128、81373140）;全军医学科技“十二五”科研项目面上项目（CWS11J097）;全军后勤科研计划重点项目（BWS14J048）

摘　　要：目的观察腺病毒介导的人EGF（Ad-hEGF）基因转染人表皮细胞的合适条件及转染对该细胞生物学特性的影响。方法取包皮环切术后弃用的新鲜人体包皮组织，通过酶消化法分离人表皮细胞，传代培养，采用第3代细胞进行以下实验。（1）按随机数字表法（分组方法下同）将细胞分为未转染组及5、20、50、100、150、200倍转染组，每组3孔。未转染组不转染Ad-hEGF基因，后6组分别按感染复数（MOI）5、20、50、100、150、200转染Ad-hEGF基因。转染24、48、72h，倒置相差显做镜下观察细胞形态，倒置荧光显微镜下观察细胞绿色荧光蛋白表达。（2）另取3个批次细胞，分别同前分组处理，分别于转染24、48、72h，流式细胞仪检测Ad-hEGF基因转染率（样本数为3），ELISA法检测收集的细胞培养上清液中EGF质量浓度（样本数为6），细胞计数试剂盒8（CCK8）及酶标仪检测细胞增殖活性（样本数为6）。（3）另取细胞分为未转染组和转染组，每组6孔。未转染组用未转染细胞的细胞培养上清液孵育，转染组用以最佳MOI（50）转染Ad-hEGF基因细胞的细胞培养上清液孵育，分别于孵育1、3、5d，CCK8及酶标仪检测细胞分泌的EGF生物活性。（4）另取细胞分为未转染组和转染组，每组12孔。未转染组不转染Ad-hEGF基因，转染组以最佳MOI（50）转染Ad-hEGF基因。转染24h，免疫荧光染色法检测细胞角蛋白14（CK14）、CK19表达。（5）另取细胞，同（4）分组（每组6孔）处理，于划痕后0（即刻）、12、24、48h，倒置相差显微镜下观测细胞迁移距离。对数据行析因设计方差分析、重复测量方差分析、LSD检验。结果（1）转染24、48h，各转染组细胞形态与未转染组比较，无明显改变；转染72h，200倍转染组的细胞碎片较未转染组明显增多。转染24、48、72h，未转染组细胞未见绿色荧光蛋白表达，各转染组Objective To investigate the suitable transfection condition of human epidermal cells （hECs） with human epidermal growth factor （EGF） gene by adenovirus vector （Ad-hEGF） and its effects on the biological characteristics of hECs. Methods hECs were isolated from deprecated human flesh pre- puce tissue of circumcision by enzyme digestion method and then sub-cultured, hECs of the third passage were used in the following experiments. （ 1 ） Cells were divided into non-transfection group and 5, 20, 50, 100, 150, and 200 fold transfection groups according to the random number table （the same grouping meth- od below） , with 3 wells in each group. Cells in non-transfeetion group were not transfected with Ad-hEGF gene, while cells in the latter six groups were transfected with Ad-hEGF gene in multiplicities of infection （MOI） of 5, 20, 50, 100, 150, and 200 respectively. The morphology of the cells was observed with in- verted phase contrast microscope, and expression of green fluorescent protein of the cells was observed with inverted fluorescence microscope at transfection hour （TH） 24, 48, and 72. （2） Another three batches of cells were collected, grouped, and treated as above, respectively. Then the transfection rate of Ad-hEGF gene was detected by flow cytometer （ n = 3） , the mass concentration of EGF in culture supernatant of cells was detected by enzyme-linked immunosorbent assay （ n = 6） , and the proliferation activity of cells was de- tected by cell counting kit 8 （CCK8） and microplate reader （ n = 6） at TH 24, 48, and 72, respectively. （3） Cells were collected and divided into non-transfeetion group and transfeetion group, with 6 wells in each group. Cells in non-transfection group were cultured with culture supernatant of cells without transfection, while cells in transfection group were cultured with culture supernatant of cells which were transfected with Ad-hEGF gene in the optimum MOI （50）. CCK8 and mieroplate reader were used to measure the biological

关 键 词：表皮生长因子 转染 细胞增殖 细胞运动 人表皮细胞 生物学特性 

分 类 号：R751[医药卫生—皮肤病学与性病学]