HTRA1基因突变对血管平滑肌细胞功能的影响及其与氧化还原反应关系研究

发布时间：2018-09-13 11:49

【摘要】：CARASIL是一种直接影响脑小血管的单基因疾病,是由编码Htr A丝氨酸肽酶/蛋白酶1(HTRA1)的HTRA1基因突变引起。CARASIL在成年早期发病,多发生于男性,其确切发病率尚不清楚。主要临床表现为缺血性脑卒中或逐步恶化的脑功能、痴呆、早秃和严重腰痛、变形性脊椎病和椎间盘突出等。影像学特征表现是通过头部CT扫描和脑磁共振成像观察到位于基底节和丘脑的弥漫性白质改变、多发腔隙性脑梗死。在组织病理学上,CARASIL主要表现为小穿通动脉的动脉硬化伴随着内膜增厚、血管平滑肌细胞损失和中膜透明变性。HTRA1基因位于10q26区域,包含有9个外显子,CARASIL相关的等位基因变异体出现在外显子1、3、4和6上,共有11个突变位点,包括7个错义突变、1个移码突变、2个无义突变和1个缺失突变。HTRA1是一种分泌型丝氨酸蛋白酶,可以促进细胞外基质蛋白的变性,也起着重要的生理作用,与关节炎、癌症疾病、家族性缺血性脑小血管病、年龄相关性黄斑变性和阿尔茨海默氏病等有关。HTRA1蛋白酶功能丧失导致CARASIL,但确切的机制尚不完全明确。2007年,我们报道了国内首例CARASIL家系,经基因检测分析,发现了在外显子6上的一个新的HTRA1基因错义突变:1091TC(点突变)。前期的研究发现,该位点突变的HTRA1基因可以导致HTRA1m RNA及其蛋白的表达减少,并引起TGF-β1/Smads/CTGF信号通路的表达上调。在本实验中,我们利用HTRA突变型和野生型基因慢病毒表达载体感染人脑血管平滑肌细胞,观察该细胞的功能及氧化还原反应情况,以此来探讨CARASIL的发病机制。第一部分HTRA1基因过表达慢病毒载体构建研究目的:构建针对HTRA1基因以及其1091TC突变基因(HTRA1-Mut)的过表达慢病毒载体。研究方法:利用NCBI Genbank数据库设计并构建HTRA1基因的引物,利用PCR法钓取并扩增野生型和突变型HTRA1基因并酶切慢病毒载体GV287,将HTRA1基因重组入GV287并转化预先培养好的感受态细胞。挑选克隆行PCR初步鉴定HTRA1重组的情况,对PCR鉴定阳性的细胞送基因测序,与Genbank数据库中的HTRA1序列进行比对,验证慢病毒载体构建成功。将制备好的GV287载体同两种装载慢病毒辅助原件的载体抽提后共同转染293T细胞系,并用western blot进行检测转入的HTRA1基因表达情况,荧光法和Elisa法检测病毒滴度。结果:通过设计的引物PCR扩增得到HTRA1基因以及HTRA1-Mut基因的目的片段,同GV287进行酶切后成功转化细菌感受态细胞,PCR鉴定阳性的克隆进行测序和比对分析显示与Genbank数据库中的HTRA1基因以及HTRA1-Mut基因序列一致。HTRA1/HTRA1-Mut表达载体转染293T后,细胞内可观察到明显的荧光,测定病毒滴度为:2E+8TU/ml。结论:成功构建了HTRA1基因及HTRA1-Mut基因真核表达载体;成功对HTRA1及HTRA1-Mut慢病毒进行了包装与滴度检测。第二部分HTRA1基因慢病毒载体感染血管平滑肌细胞模型的建立研究目的:对人脑血管平滑肌细胞(HBVSMC)进行HTRA1及HTRA1-Mut过表达慢病毒载体感染。研究方法:对HBVSMC进行细胞培养,α-SMA抗体荧光染色进行表型鉴定;对培养后生长良好的HBVSMC进行HTRA1及HTRA1-Mut过表达慢病毒载体感染。结果:HBVSMC生长状态良好,光镜下细胞形态正常,对细胞行,α-SMA抗体染色细胞荧光显色良好;HTRA1及HTRA1-Mut过表达慢病毒载体感染后HBVSMC有荧光表达,并且荧光率达80%以上。结论:成功培养HBVSMC细胞系并进行了初步鉴定成功;成功建立HTRA1及HTRA1-Mut基因慢病毒载体感染血管平滑肌细胞模型。第三部分HTRA1基因突变对人脑血管平滑肌细胞的增殖、迁移、凋亡的影响研究目的:检测HTRA1及HTRA1-Mut基因慢病毒载体转染HBVSMC后,HBVSMC的增殖、迁移及凋亡的变化。研究方法:将HBVSMC分为三组,NC正常的人脑血管平滑肌细胞组,OE-WT HTRA1野生型病毒感染细胞组及OE-MU HTRA1突变型病毒感染细胞组,细胞培养良好后分别行CCK-8检测细胞增殖,Transwell实验检测细胞的迁移,细胞凋亡用流式细胞术检测。结果:相比正常的人脑血管平滑肌细胞(NC),HTRA1野生型病毒感染细胞(OE-WT)增殖无明显变化,HTRA1野生型病毒感染细胞(OE-MU)增殖减缓。与正常的人脑血管平滑肌细胞(NC)相比,HTRA1野生型病毒感染细胞(OE-WT)Transwell转移率无明显差异(P0.05),HTRA1突变型病毒感染细胞(OE-MU)Transwell转移率降低(P0.05);OE-WT组与OE-MU组相比,OE-MU组的人脑血管平滑肌细胞的转移率明显降低(P0.05)。与正常细胞组(NC)相比,HTRA1野生型病毒感染细胞(OE-WT)凋亡数减少(p0.05),而在HTRA1突变型病毒感染细胞(OE-MU)未见明显凋亡数减少(P0.05),与HTRA1野生型病毒感染细胞(OE-WT)相比,HTRA1突变型病毒感染细胞(OE-MU)凋亡数增多(P0.05)。结论:HTRA1突变型基因感染人脑血管平滑肌细胞后引起该细胞增殖能力和迁移活性减弱,并且影响细胞凋亡。第四部分HTRA1基因对突变血管平滑肌细胞氧化反应的影响研究目的: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。研究方法: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Real-time PCR和Western Blot的方法检测三组细胞的NOXm RNA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。结果: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NOX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LV-HRTA1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。结论:1、HTRA1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,NOX4m RNA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它两组均升高。2、HTRA1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。
[Abstract]:CARASIL is a monogenic disease that directly affects the small blood vessels of the brain. It is caused by a mutation in the Htr A serine peptidase/protease 1 (HTRA1) gene. CARASIL occurs in early adulthood, mostly in men, and its exact incidence is not yet known. The main clinical manifestations are ischemic stroke or progressive deterioration of brain function, dementia, early alopecia and premature alopecia. Severe low back pain, deformable spondylopathy, and intervertebral disc herniation. Imaging features include diffuse white matter changes in the basal ganglia and thalamus, and multiple lacunar infarctions. Histopathologically, CARASIL is characterized by arteriosclerosis of small perforating arteries with intimal thickening. Vascular smooth muscle cell loss and clear degeneration of the mesangium. HTRA1 gene is located in the 10q26 region and contains nine exons. CARASIL-related allele variants appear on exons 1, 3, 4 and 6. There are 11 mutations, including 7 missense mutations, 1 shift mutation, 2 nonsense mutations and 1 deletion mutation mutation. HTRA1 is a secretory serine. Acid protease, which can promote the degeneration of extracellular matrix protein, also plays an important physiological role. It is related to arthritis, cancer, familial ischemic cerebellar vascular disease, age-related macular degeneration and Alzheimer's disease. Loss of function of HTRA1 protease leads to CARASIL, but the exact mechanism is not yet clear. A new missense mutation of HTRA1 gene on exon 6, 1091TC (point mutation), was found in the first CARASIL family in China. Previous studies have shown that the mutation of HTRA1 gene can reduce the expression of HTRA1m RNA and its protein, and induce the up-regulation of TGF-beta 1/Smads/CTGF signaling pathway. In this study, we used HTRA mutant and wild-type lentiviral expression vectors to infect human cerebral vascular smooth muscle cells, and observed the function and redox reaction of the cells, so as to explore the pathogenesis of CARASIL. Methods: The primers of HTRA1 gene were designed and constructed using NCBI Genbank database. Wild and mutant HTRA1 genes were harvested and amplified by PCR and the lentiviral vector GV287 was digested. The HTRA1 gene was recombined into GV287 and transformed into pre-cultured competent cells. The recombinant HTRA1 was preliminarily identified by PCR, and the positive cells identified by PCR were sequenced and compared with the HTRA1 sequence in Genbank database to verify the success of the construction of lentiviral vector. Results: The target fragments of HTRA1 gene and HTRA1-Mut gene were amplified by designed primers PCR. After digestion with GV287, the transfected cells were transformed successfully. The positive clones identified by PCR were sequenced and compared with Genbank number. The sequence of HTRA1 gene and HTRA1-Mut gene in the database was identical. After transfection of 293T with HTRA1/HTRA1-Mut expression vector, obvious fluorescence was observed and the viral titer was determined as 2E+8TU/ml. Conclusion: Eukaryotic expression vectors of HTRA1 gene and HTRA1-Mut gene were successfully constructed, and HTRA1 and HTRA1-Mut lentiviruses were successfully packaged and titered. Objective: To establish a model of human cerebral vascular smooth muscle cells (HBVSMC) infected with HTRA1 and HTRA1-Mut lentiviral vectors. Methods: HBVSMC cells were cultured and phenotyped by alpha-SMA antibody fluorescence staining. HBVSMC was infected by lentiviral vector with over-expression of HTRA1 and HTRA1-Mut. Results: HBVSMC grew well, the morphology of cells was normal under light microscope, and the cells stained with anti-alpha-SMA antibody showed good fluorescence staining; HBVSMC with over-expression of HTRA1 and HTRA1-Mut expressed fluorescence, and the fluorescence rate was over 80%. BVSMC cell lines were identified successfully and the model of VSMC infected with HTRA1 and HTRA1-Mut lentiviral vector was established successfully. Part III The effect of HTRA1 gene mutation on proliferation, migration and apoptosis of human VSMC Objective: To detect the effect of HTRA1 and HTRA1-Mut lentiviral vector on HBVSMC transfection. Methods: HBVSMC was divided into three groups: normal NC human cerebral vascular smooth muscle cell group, OE-WT HTRA1 wild type virus infection cell group and OE-MU HTRA1 mutant virus infection cell group. Results: Compared with normal human cerebrovascular smooth muscle cells (NC), the proliferation of HTRA1 wild-type virus-infected cells (OE-WT) was not significantly changed, and the proliferation of HTRA1 wild-type virus-infected cells (OE-MU) was slowed down. Compared with normal human cerebrovascular smooth muscle cells (NC), the Transwell metastasis of HTRA1 wild-type virus-infected cells (OE-WT) was observed. There was no significant difference (P 0.05), but the Transwell metastasis rate of HTRA1 mutant virus-infected cells (OE-MU) was decreased (P 0.05). Compared with OE-MU group, the metastasis rate of human cerebral vascular smooth muscle cells in OE-WT group was significantly decreased (P 0.05). Compared with NC group, the apoptosis of HTRA1 wild type virus-infected cells (OE-WT) was decreased (P 0.05), but the apoptosis rate of HTRA1 protrusion was decreased (P 0.05). There was no significant decrease in the number of apoptosis in OE-MU cells (P 0.05). Compared with OE-WT cells infected by HTRA1, the number of apoptosis in OE-MU cells infected by HTRA1 was increased (P 0.05). Conclusion: The proliferation and migration activity of OE-MU cells infected by HTRA1 mutation gene were decreased, and the migration activity was also decreased. Objective: To detect the changes of oxidative stress in HBVSMC transfected with HTRA1 and HTRA1-Mut lentiviral vectors. Methods: NC and OE were collected at specific time points after HTRA1 and HTRA1-Mut lentiviral vectors were transfected into HBVSMC. The expression of NOXm RNA and protein in three groups of cells was detected by Real-time PCR and Western Blot respectively, and the levels of reactive oxygen species (ROS) in three groups of cells were detected by DCFH-DA method. The expression level of NOX protein in normal human cerebral vascular smooth muscle cells was lower than that in normal human cerebral vascular smooth muscle cells, but the expression level of NOX 4 protein was higher after infection with lentivirus LV-HRTA1 and LV-HRTA1-MUT. The expression of ROS in normal human cerebral vascular smooth muscle cells was lower than that in lentivirus LV-HRTA1-MUT cells. CONCLUSION: 1. The production of reactive oxygen species (ROS) and the expression of NOX4m RNA in human cerebral vascular smooth muscle cells infected with HTRA1 mutant gene increased, but NOX4 was not significantly different from that of HTRA1 wild type gene. Compared with the other two groups, the expression of protein was increased. 2. The proliferation, migration and apoptosis of human cerebral vascular smooth muscle cells infected with HTRA1 mutant gene decreased, which may be related to oxidative stress in cells.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R743

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