腺病毒介导的人肝细胞生长因子对大鼠脑膜间皮细胞的转染效率及增殖和迁移效应

Transfection efficiency of adenoviral vector expressing human hepatocyte growth factor with rat meningeal mesothelial cells in vitro and its effect on proliferation and migration

目的观察腺病毒介导的人肝细胞生长因子(Ad-HGF)对大鼠脑膜间皮细胞(RMMCs)的转染效率及增殖和迁移效应。方法原代培养RMMCs并鉴定;携带增强型GFP基因的腺病毒载体(Ad5-EGFP)转染RMMCs,通过荧光显微镜和流式细胞仪检测其转染细胞的最佳转染强度(MOI);酶联免疫吸附(ELISA)法检测不同转染强度(25、50、100及200 pfu/细胞)Ad-HGF转染RMMCs后上清液中HGF的表达;ELISA法检测不同时间点(0、2、4、6、8、10、12及14 d)上清液中HGF的表达;MTT法检测不同体积百分数(0%、25%、50%、75%、100%)表达上清对细胞的增殖效应;Transwells法和划痕实验检测表达上清对细胞的迁移效应。所有数据均以(x珋±s)表示,统计学处理由SPSS 13.0软件完成,采用曲线拟合完成直线回归方程,两样本均数比较用独立样本t检验,多组间比较选用单因素方差分析,α=0.05为标准检验水准。结果体外成功培养并鉴定RMMCs;Ad5-EGFP转染细胞MOI为200 pfu/细胞,转染效率高达96.21%;上清液中HGF的表达随MOI增加而增加,且呈一定浓度依赖性,差异有显著性(F=407.851,P<0.01),MOI=200 pfu/细胞时表达量为(97.275±3.035)ng/ml;HGF表达量呈一定时间依赖性,差异有统计学意义(F=384.063,P<0.01),转染后48 h达高峰,峰值为(104.275±2.356)ng/ml,随后下降,10 d下降明显,随后保持相对稳定水平;表达上清对RMMCs有明显促增殖效应,且呈一定浓度依赖性,差异有统计学意义(F=285.946,P<0.01);表达上清对RMMCs有促迁移效应,差异有统计学意义(t=34.135,P<0.01)。结论 Ad-HGF可高效转染大鼠脑膜间皮细胞并在细胞中表达,表达上清对细胞有明显促增殖和促迁移效应。

Objective To investigate the transfection efficiency of adenoviral vector expressing human hepatocyte growth factor （Ad-HGF） with rat meningeal mesothelial cells （RMMC） in vitro and its effect on proliferation and migration. Methods RMMC were primary cultured and identified in vitro. Transfection efficiency was detected by fluorescence microscope and flow cytometer. The expression of HGF was measured with enzyme linked immunosorbent assay（ELISA） at different MOI of Ad-HGF or time points. The effect on proliferation of HGF was evaluated with MTY assay. The effect on migration of HGF was detected with Scratch wounds experiment and Transwells assay. All data were indicated by mean ＋ standard deviation and put to SPSS 13.0 sofaware. The linear regression equation was made through curve fitting analysis, t-test was applied to compare the data of two groups, and one-way ANOVA analysis was used to compare the data of groups. The standard test level was a = 0.05. Results RMMC were successfully isolated, culture-expanded and identified in vitro, best MOI of adenovirus5 vector encoding enhanced GFP gene （AdS-EGFP） was 200 that the transfection efficiency was 96.21%. The expression of HGF in the supernatant was increasing along with the increasing of MOI in a dose-dependent manner （ F = 407. 851, P 〈 0. 05 ）, and the highest concentration was （97. 275 ± 3. 035 ） ng./ml at 200 PFU/eell. The expression of HGF was in a time-dependent manner（ F = 384. 063, P 〈 0.05）, which became the peak of （104. 275 ±2. 356）ng/ml after 48 h and sharply decreased on the 10th day, then sustained at a stable level. The supernatant promoted the effect of proliferation in a does-dependent manner （ F = 285. 946, P 〈 0.05 ）. The supernatant promoted the effect of migration compared with control group （ t = 34. 135, P 〈 0. 05 ）. Conclusion Ad-HGF can transfeet efficiently RMMC and the transfected cells can express the aim protein efficiently. HGF can promote the effects of proliferation and migration of RMMC.