【摘要】：CARASIL是一種直接影響腦小血管的單基因疾病,是由編碼Htr A絲氨酸肽酶/蛋白酶1(HTRA1)的HTRA1基因突變引起。CARASIL在成年早期發(fā)病,多發(fā)生于男性,其確切發(fā)病率尚不清楚。主要臨床表現(xiàn)為缺血性腦卒中或逐步惡化的腦功能、癡呆、早禿和嚴重腰痛、變形性脊椎病和椎間盤突出等。影像學特征表現(xiàn)是通過頭部CT掃描和腦磁共振成像觀察到位于基底節(jié)和丘腦的彌漫性白質改變、多發(fā)腔隙性腦梗死。在組織病理學上,CARASIL主要表現(xiàn)為小穿通動脈的動脈硬化伴隨著內膜增厚、血管平滑肌細胞損失和中膜透明變性。HTRA1基因位于10q26區(qū)域,包含有9個外顯子,CARASIL相關的等位基因變異體出現(xiàn)在外顯子1、3、4和6上,共有11個突變位點,包括7個錯義突變、1個移碼突變、2個無義突變和1個缺失突變。HTRA1是一種分泌型絲氨酸蛋白酶,可以促進細胞外基質蛋白的變性,也起著重要的生理作用,與關節(jié)炎、癌癥疾病、家族性缺血性腦小血管病、年齡相關性黃斑變性和阿爾茨海默氏病等有關。HTRA1蛋白酶功能喪失導致CARASIL,但確切的機制尚不完全明確。2007年,我們報道了國內首例CARASIL家系,經基因檢測分析,發(fā)現(xiàn)了在外顯子6上的一個新的HTRA1基因錯義突變:1091TC(點突變)。前期的研究發(fā)現(xiàn),該位點突變的HTRA1基因可以導致HTRA1m RNA及其蛋白的表達減少,并引起TGF-β1/Smads/CTGF信號通路的表達上調。在本實驗中,我們利用HTRA突變型和野生型基因慢病毒表達載體感染人腦血管平滑肌細胞,觀察該細胞的功能及氧化還原反應情況,以此來探討CARASIL的發(fā)病機制。第一部分HTRA1基因過表達慢病毒載體構建研究目的:構建針對HTRA1基因以及其1091TC突變基因(HTRA1-Mut)的過表達慢病毒載體。研究方法:利用NCBI Genbank數據庫設計并構建HTRA1基因的引物,利用PCR法釣取并擴增野生型和突變型HTRA1基因并酶切慢病毒載體GV287,將HTRA1基因重組入GV287并轉化預先培養(yǎng)好的感受態(tài)細胞。挑選克隆行PCR初步鑒定HTRA1重組的情況,對PCR鑒定陽性的細胞送基因測序,與Genbank數據庫中的HTRA1序列進行比對,驗證慢病毒載體構建成功。將制備好的GV287載體同兩種裝載慢病毒輔助原件的載體抽提后共同轉染293T細胞系,并用western blot進行檢測轉入的HTRA1基因表達情況,熒光法和Elisa法檢測病毒滴度。結果:通過設計的引物PCR擴增得到HTRA1基因以及HTRA1-Mut基因的目的片段,同GV287進行酶切后成功轉化細菌感受態(tài)細胞,PCR鑒定陽性的克隆進行測序和比對分析顯示與Genbank數據庫中的HTRA1基因以及HTRA1-Mut基因序列一致。HTRA1/HTRA1-Mut表達載體轉染293T后,細胞內可觀察到明顯的熒光,測定病毒滴度為:2E+8TU/ml。結論:成功構建了HTRA1基因及HTRA1-Mut基因真核表達載體;成功對HTRA1及HTRA1-Mut慢病毒進行了包裝與滴度檢測。第二部分HTRA1基因慢病毒載體感染血管平滑肌細胞模型的建立研究目的:對人腦血管平滑肌細胞(HBVSMC)進行HTRA1及HTRA1-Mut過表達慢病毒載體感染。研究方法:對HBVSMC進行細胞培養(yǎng),α-SMA抗體熒光染色進行表型鑒定;對培養(yǎng)后生長良好的HBVSMC進行HTRA1及HTRA1-Mut過表達慢病毒載體感染。結果:HBVSMC生長狀態(tài)良好,光鏡下細胞形態(tài)正常,對細胞行,α-SMA抗體染色細胞熒光顯色良好;HTRA1及HTRA1-Mut過表達慢病毒載體感染后HBVSMC有熒光表達,并且熒光率達80%以上。結論:成功培養(yǎng)HBVSMC細胞系并進行了初步鑒定成功;成功建立HTRA1及HTRA1-Mut基因慢病毒載體感染血管平滑肌細胞模型。第三部分HTRA1基因突變對人腦血管平滑肌細胞的增殖、遷移、凋亡的影響研究目的:檢測HTRA1及HTRA1-Mut基因慢病毒載體轉染HBVSMC后,HBVSMC的增殖、遷移及凋亡的變化。研究方法:將HBVSMC分為三組,NC正常的人腦血管平滑肌細胞組,OE-WT HTRA1野生型病毒感染細胞組及OE-MU HTRA1突變型病毒感染細胞組,細胞培養(yǎng)良好后分別行CCK-8檢測細胞增殖,Transwell實驗檢測細胞的遷移,細胞凋亡用流式細胞術檢測。結果:相比正常的人腦血管平滑肌細胞(NC),HTRA1野生型病毒感染細胞(OE-WT)增殖無明顯變化,HTRA1野生型病毒感染細胞(OE-MU)增殖減緩。與正常的人腦血管平滑肌細胞(NC)相比,HTRA1野生型病毒感染細胞(OE-WT)Transwell轉移率無明顯差異(P0.05),HTRA1突變型病毒感染細胞(OE-MU)Transwell轉移率降低(P0.05);OE-WT組與OE-MU組相比,OE-MU組的人腦血管平滑肌細胞的轉移率明顯降低(P0.05)。與正常細胞組(NC)相比,HTRA1野生型病毒感染細胞(OE-WT)凋亡數減少(p0.05),而在HTRA1突變型病毒感染細胞(OE-MU)未見明顯凋亡數減少(P0.05),與HTRA1野生型病毒感染細胞(OE-WT)相比,HTRA1突變型病毒感染細胞(OE-MU)凋亡數增多(P0.05)。結論:HTRA1突變型基因感染人腦血管平滑肌細胞后引起該細胞增殖能力和遷移活性減弱,并且影響細胞凋亡。第四部分HTRA1基因對突變血管平滑肌細胞氧化反應的影響研究目的:檢測HTRA1及HTRA1-Mut基因慢病毒載體轉染HBVSMC后,HBVSMC內氧化應激水平的變化。研究方法:HTRA1及HTRA1-Mut基因慢病毒載體轉染HBVSMC后,在特定的時間點收集NC、OE-WT HTRA1及OE-MU HTRA1三組細胞的總RNA及總蛋白,分別用Real-time PCR和Western Blot的方法檢測三組細胞的NOXm RNA及蛋白水平的表達情況,用DCFH-DA法檢測三組細胞內的活性氧水平。結果:從定量PCR結果可以看出,人腦血管平滑肌細胞中,OE-MU組NOX4基因表達豐度是NC組的2.015倍。從Western blot結果可以看出,在正常的人腦血管平滑肌細胞中NOX蛋白水平表達較低,而在慢病毒LV-HRTA1及LV-HRTA1-MUT感染后NOX4蛋白表達水平增高。在正常的人腦血管平滑肌細胞中ROS水平表達較低,而在慢病毒LV-HRTA1及LV-HRTA1-MUT感染后ROS蛋白表達水平增高,在突變型病毒感染細胞組表現(xiàn)更為明顯。結論:1、HTRA1突變型基因感染人腦血管平滑肌細胞后,細胞內活性氧產量增加,NOX4m RNA水平的表達正常細胞升高,但較HTRA1野生型基因無明顯差別;NOX4在蛋白水平表達較其它兩組均升高。2、HTRA1突變型基因感染的人腦血管平滑肌細胞出現(xiàn)增殖減少、遷移活力降低以及凋亡增加可能與細胞內的氧化應激有關,為進一步研究CARASIL發(fā)病機制奠定基礎。
[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.
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R743

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