慢病毒介导的HTRA-突变基因感染对人脑血管平滑肌细胞生物学特性影响

Effect of high temperature requirement protease AI lentivirus expression vector infection onbiological behavior of human vascular smooth muscle cells

目的探讨HTRAJ基因及tfTRA，突变基因[1091T〉C（点突变）]慢病毒表达载体感染对人脑血管平滑肌细胞（HBVSMC）增殖、迁移及凋亡等生物学特性的影响。方法构建野生型HTRAJ基因及突变型HTRAJ基因慢病毒表达载体，并将其感染经培养和鉴定的HBVSMC，以空载体感染libVSMC为对照组。采用CCK-8法连续5d检测细胞增殖情况，Transwell实验检测细胞的迁移，流式细胞术检测细胞凋亡。结果与对照组相比，野生型HTRAI基因慢病毒组细胞增殖无明显变化，至第3天起突变型HTRAl基因慢病毒组细胞增殖减缓，差异具有统计学意义（P〈0．05）。对照组、野生型HTRAl基因慢病毒组、突变型HTRAI基因慢病毒组细胞迁移率分别为0．474±0．079、0．612±0．037、0．283±0．064，差异有统计学意义（p〈0．05）。与对照组细胞凋亡率（3．68％±0.23％）相比，野生型HTRAI基因慢病毒组细胞凋亡率（3．13％±0．07％1降低，差异有统计学意义（P〈0．05）；突变型HTRAI基因慢病毒组细胞凋亡率（3.70％±0.20％）增多不明显，差异无统计学意义（P〉0．05）。结论突变型HTRAI基因感染HBVSMC后引起该细胞增殖能力和迁移活性减弱，但对细胞凋亡无影响。

Objective To construct the lentivirus vectors of high temperature requirement protease A1 （HTRA1） gene as well as it＇s mutant gene （1091 T〉C HTRA1）, and investigate the effect of their infection on proliferation, migration and apoptosis of human brain vascular smooth muscle cells （HBVSMC）. Methods The lentivirus vectors of wild type HTRA1 and mutant HTRA1 were constructed, cultured and identified, and then, infected into HBVSMC. Empty lentivirus vectors were used as controls. CCK-8 method was used to detect the cell proliferation for a consecutive five d. Transwell assay was used to detect the cell migration, and cell apoptosis was detected by flow cytometry. Results As compared with those from the control group, cells fi＇om wild-type HTRA 1 group showed no statistically different proliferation rate （P〉0.05）, while cells from mutant HTRA 1 group had significantly reduced proliferation rate since the third d （P〈0.05）. Migration rate of cells from control group, wild type HTRA 1 group and mutant HTRA 1 group was 0.474±0.079, 0.612±0.037, and 0.283±0.064, respectively, with significant differences （P〈0.05）. Apoptosis percentage of cells from control group, wild type HTRA 1 group was 3.68%±0.23% and 3.13%±0.07%, with significant difference （P〈0.05）, while that of mutant H TRA 1 groupwas3.70%±02.0%, showingnosignificantdifferenceascomparedwiththatfromcontrolgroup（P〈0.05）. Conclusion HBVSMC infected by HTRA 1 mutant gene display attenuation of proliferation and migration activity, but have no statistical change in apoptosis.