慢病毒介导的HTRA1突变基因感染对人脑血管平滑肌细胞内氧化应激反应的影响

检测HTRA1及HTRA1-Mut基因慢病毒载体转染HBVSMC后,HBVSMC内氧化应激水平的变化。以HTRA1及HTRA1-Mut基因慢病毒载体转染HBVSMC后,在特定的时间点收集NC、OE-WT HTRA1及OE-MU HTRA1三组细胞的总RNA及总蛋白,分别用RT-PCR和Western Blot的方法检测三组细胞的NOX4 mRNA及蛋白水平的表达情况;用DCFH-DA法检测三组细胞内的活性氧水平。从定量PCR结果可以看出,人脑血管平滑肌细胞中,OE-MU组NOX4基因表达丰度是NC组的2.015倍。从Western blot结果可以看出,在正常的人脑血管平滑肌细胞中NOX4蛋白水平表达较低,而在慢病毒LV-HRTA1及LV-HRTA1-MUT感染后NOX4蛋白表达水平增高。在正常的人脑血管平滑肌细胞中ROS水平表达较低,而在慢病毒LVHRTA1及LV-HRTA1-MUT感染后ROS蛋白表达水平增高,在突变型病毒感染细胞组表现更为明显。说明:1HTRA1突变型基因感染人脑血管平滑肌细胞后,细胞内活性氧产量增加,NOX4 mRNA水平的表达正常细胞升高,但较HTRA1野生型基因无明显差别;NOX4在蛋白水平表达较其他两组均升高;2HTRA1突变型基因感染的人脑血管平滑肌细胞出现增殖减少、迁移活力降低以及凋亡增加可能与细胞内的氧化应激有关,为进一步研究CARASIL发病机制奠定基础。

Effect of HTRA1 gene mutation on oxidation reactionof human brain vascular smooth muscle cells

Objective: Detection of changes on oxidative stress levels of HBVSMCs after infected by HTRA1 wild and HTRA1 mutant gene lentivirus vectors. Methods: HBVSMCs which were infected by HTRA1 wild gene and HTRA1 mutant gene lentivirus vectors were divided into three groups, NC group, OE-WT group and OE-MU group. DCFH-DA method was used to detect the levels of intracellular reactive oxygen species in three groups. The total RNA and total protein of three groups of cells were collected at a given time, and detected expression level of NOX4 mRNA and NOX4 protein by Real-time PCR and Western Blot. Results: The level of ROS in normal HBVSMCs was low, while increased after infection of HTRA1 wild gene and HTRA1 mutant gene lentivirus vectors. It was more obvious in the mutant lentivirus infected cells. In the part of Rt-PCR, NOX4 gene expression abundance in OE-MU group was 2.015 times higher than NC group. About Western blot, the level of NOX4 protein expression in normal human vascular smooth muscle cells was low, while was higher in HTRA1 wild gene and HTRA1 mutant gene infected HBVSMCs. Conclusions: Intracellular ROS production of HBVSMCs was increased after infected by HTRA1 mutant gene lentivirus vector. The expression level of NOX4 mRNA in HTRA1 mutant gene cells had no significant difference compared with the wile type of HTRA1 gene. The expression level of NOX4 protein was higher than the other two groups.