【作者】 王晓玲；

【导师】 曹秉振；

【作者基本信息】 第二军医大学 ， 神经病学， 2012， 博士

【摘要】 伴有皮质下梗死和白质病变的常染色体隐性遗传性脑动脉病（cerebral autosomalrecessive arteriopathy with subcortical infarcts and leucoencephalopathy，CARASIL）是一种隐性遗传性脑血管病，以青年早发的痴呆、卒中、腰痛、秃顶为主要临床表现；头颅影像学检查表现为皮质下白质广泛的脱髓鞘改变伴有多发腔隙性脑梗死；脑部尸检观察到脑部小动脉内膜增厚、内弹力层断裂、中膜平滑肌细胞减少、玻璃样变及管腔向心性狭窄等类似小动脉硬化的表现。半数以上病例有父母近亲结婚的家族史。整个病程平均10年。自1965年日本就有病例首报，但由于对疾病的认识不足以及诊断手段的不完善，此后仅有陆续报道，包括几个家系和一些散发病例。目前仅有50余例报道，大部分来自日本。2009年日本学者汇总了5个家系，通过基因连锁分析的方法将该病的致病基因定位于10号染色体的HTRA1基因。该基因有9个外显子，目前报道的突变位点包括4个错义突变，2个无义突变，它们分布在第3、4、6外显子上。这一区域位于HtrA1丝氨酸蛋白酶的活性区，所以往往会导致酶活性改变。但关于这方面的实验研究结论十分缺乏。HtrA1丝氨酸蛋白酶在体内广泛存在并且作用于多种靶蛋白，大部分为细胞外基质成分，与人类骨性关节炎、肿瘤等疾病有关。与HTRA1基因有关的信号通路为TGF-β/Smads，HtrA1丝氨酸蛋白酶能够抑制该信号通路，而基因突变则使蛋白酶活性减低，TGF-β/Smads信号通路表达上调，包括TGF-β1、BMP-2、BMP-4蛋白，从而通过不同途径导致CARASIL脑小动脉的病变和神经系统以外的症状。由于病理上能够见到比较明显的动脉中膜平滑肌细胞的大量丢失，所以研究血管平滑肌细胞的结构和功能变化对于进一步探讨CARASIL的发病机制尤为重要。目前国内外尚无HTRA1基因转染平滑肌细胞的相关研究。2007年我们在国内首次临床报道了一CARASIL家系，本课题旨在研究这一家系HTRA1基因突变方式，并建立一个HTRA1野生型及突变型基因的平滑肌细胞表达模型，检测TGF-β1/Smads信号通路分子的变化，有助于进一步理解CARASIL血管病变的病理机制。第一部分CARASIL一家系的临床、病理和基因分析研究目的：探讨一个CARASIL家系的临床病理特征，并对致病基因进行分析。研究方法：描述2例患者的临床、影像及病理特征，对第l9号染色体NOTCH3基因的1-33外显子全段测序，第l0号染色体HTRA1基因9个外显子测序。同时设正常人群对照进行HTRA1基因检测。结果：该家系包括2例患者，系同胞姐弟，父母近亲结婚。两例患者的发病年龄20-25岁，脑电图呈弥漫性慢波表现，头颅MRI检查均显示双侧大脑半球弥漫性白质病变，伴有皮质下多发性梗死。腓肠神经活检可见小动脉内弹力层轻度分裂、中层肥厚、血管腔呈向心性狭窄，PAS染色未见颗粒状沉积物，淀粉染色阴性，电镜下在小动脉平滑肌细胞层没有发现嗜锇颗粒。对第l9号染色体NOTCH3基因的1-33外显子全段测序未观察到突变。对HTRA1基因第1-9外显子测序发现第6外显子1091T>C纯合错义突变（L364P），患者父母及患者1的女儿为该位点的杂合突变。对40例对照进行HTRA1基因的测序未发现该位点突变或多态性改变。结论：我们对国内第一个CARASIL家系从基因水平进行了诊断，在HTRA1基因的第6外显子发现一个新的突变位点。第二部分HTRA1基因真核表达载体转染血管平滑肌细胞模型的建立研究目的：构建针对HTRA1基因的pEGFP-N1载体以及1091T>C突变质粒载体，并将两种质粒载体转染到血管平滑肌细胞。研究方法：根据人HTRA1基因CDS区的序列设计一对引物，上游：5’–CTCAAGCTTCGAATTCATGCAGATCCCGCGCGCCGCTCTTC-3’；下游：5’-GGCGACCGGTGGATCCCG TGGG TCAATTTCTTCGGGAA-3’。PCR扩增目的基因，将目的片段与p-EGFP-N1载体连接后转化大肠杆菌，抽提质粒后用EcoR I和BamH I双酶切后琼脂糖电泳及质粒测序验证，此为野生型质粒载体（WT）。另一试验组根据我们发现的突变位点1091T>C，设计两条包含有突变位点的中间引物，应用基因定点突变原理扩增含有突变位点的两段PCR产物，并将产物与pEGFP-N1载体进行连接反应，构建突变质粒载体（L364P）。转化感受态大肠杆菌进行扩增，提取质粒并进行双酶切及测序鉴定。用SMCM培养基培养血管平滑肌细胞，用α-SM-actin抗体免疫细胞化学鉴定细胞。将VSMC随机分为三组，分别应用DNAfect转染试剂进行转染：（1）WT组：转染HTRA1-pEGFP-N1野生型质粒；（2）L364P组：转染HTRA1-pEGFP-N1突变型质粒；（3）空白对照组：转染pEGFP-N1质粒。转染后使用流式细胞仪分析转染效率。结果：（1）野生型及突变型质粒OD值在1.8-2.0之间，浓度300-400ng/ul，双酶切后可见两个条带存在，分别为4.7kbp和1400bp，符合pEGFP-N1载体和HTRA1基因的长度。最后测序鉴定正确。（2）培养的细胞为长梭形，三角形、条带型、星型等多种形态，生长密集时称典型的“峰—谷”状，单克隆α-SM-actin抗体鉴定结果阳性，证实培养的细胞为平滑肌细胞。在转染后24-48小时之内观察，三组细胞均有绿色荧光表达，表明已经成功转染。流式检测转染效率在33%。结论：成功构建HTRA1基因的野生型和突变型质粒载体。两种质粒载体采用瞬时转染的方法转染进血管平滑肌细胞，有效的建立起HTRA1突变基因血管平滑肌细胞表达模型。第三部分HTRA1基因在血管平滑肌细胞的表达研究目的：检测HTRA1野生型及突变型基因转染血管平滑肌细胞后的HTRA1mRNA及HtrA1蛋白的表达。研究方法：细胞转染48小时后，收集对照组、WT组、L364P组的细胞，提取总RNA及总蛋白，分别用荧光定量PCR及Western blot方法检测HTRA1基因的表达。结果：三组细胞内均能检测到HTRA1基因的表达。RT-PCR结果显示，与对照组相比，HTRA1mRNA在L364P组和WT组表达增强，与WT组相比，L364P组表达减少；Westernblot结果显示两个转染组的HtrA1蛋白比对照组表达增多，与WT组相比，L364P组表达减少。结论：HTRA1基因转染血管平滑肌细胞后能在细胞内成功表达。1091T>C突变后导致HTRA1基因mRNA以及HtrA1蛋白减少。第四部分HTRA1基因对血管平滑肌细胞TGF-β1/Smads信号通路的影响研究目的：检测HTRA1野生型及突变型基因转染血管平滑肌细胞后的TGF-β1/Smads信号通路的变化。研究方法：细胞转染48小时后，收集对照组、WT组、L364P组三组细胞，提取总RNA及总蛋白，分别用荧光定量PCR及Western blot方法检测TGF-β1、Smad2/3/4及磷酸化Smad2/3的表达差异。结果：荧光定量PCR结果显示，与WT组相比，L364P组中的TGF-β1及Smad2/3的mRNA表达比WT组增强，差异有统计学意义（P<0.05），smad4的表达两组差别不明显；WB结果显示Smad2/3蛋白的表达L364P组较WT组有所增加，磷酸化的Smad2/3仅在L364P组检测到。Smad4的蛋白表达L364P组较WT组略有减少，差别不明显。结论： HTRA1突变型基因(1091T>C)转染VSMC后引起TGF-β1/Smads信号通路的表达上调。更多还原

【Abstract】 Cerebral autosomal recessive arteriopathy with subcortical infarcts andleucoencephalopathy (CARASIL) is an inherited vascular diseases characterized by youngadult-onset non-hypertension stroke, progressive motor and cognitive impairment, alopeciaand lumbago. Brain MRI demonstrated diffuse leukoencephalopathy and multiple subcorticalinfarcts. Brain autopsy showed concentric thickening of vascular wall, narrowing of thelumen, mild fibrous proliferation of the intima and extensive loss of smooth muscle cells,which was similar with arteriosclerosis. About50%cases were born of the consanguineousparents. The patients would die after about10years from the onset. The first case wasreported by Japanese in1965. And then there were only a few cases reported including a fewfamilies and some sporadic cases because the understanding of the disease was insufficientand the diagnostic tools were not perfect. Up to now, only about50cases have been reportedand most of them were from Japan. In2009, some Japanese scholars collected5families ofCARASIL and found CARASIL was associated with mutations in the HTRA1gene which islocated on chromosome10q. There are9exons in HTRA1gene. To date,4missensemutations and2nonsense mutations had been reported and they were distributed in exon3,4and6where located the HtrA1protease domain. So HTRA1gene mutation might lead to thechange of its activity. But studies about that was insufficient. HtrA1serine protease waswidespread in the body and acted with a lot of target proteins, most of them were extracellularmatrix components. HtrA1serine protease was closely associated with osteoarthritis andmany types of cancers. TGF-β/Smads signaling was associated with HTRA1gene. HtrA1serine protease can inhibit the signaling. HTRA1gene mutations reduced the protease activity,and then followed with the over-expression of TGF-β/Smads, including TGF-β1、BMP-2、BMP-4. That was the cause of lesions on brain small artery and symptom of extra-nervoussystem.Extensive loss of vascular smooth muscle cells can be found pathologically, so studies onthe structure and function of vascular smooth muscle cells are important to explore thepathological mechanism of CARASIL. Up to date, there are no investigations about HTRA1gene transfection to vascular smooth muscle cells. We had reported a CARASIL family inChina for the first time. Our study was mainly on the detection of the HTRA1gene mutationsite, then we intend to build a model of wild and mutant HTRA1gene expressed in vascularsmooth muscle cells, and study on the TGF-β/Smads signaling which is helpful to explor thepathological mechanism of CARASIL. Part Ⅰ Study on the clinicalpathological and genetic characteristicof a CARASIL familyObjective: To explorer the clinicopathological characteristics of a CARASIL family, andanalyze the causative gene.Methods: The clinical, imaging and pathological characteristics were described. Thesequence of the exons from1to33on NOTCH3in chromosome19and that of exons from1to9on HTRA1gene in chromosome10were detected. Meaningwhile the healthy control wasdetected the HTRA1gene.Results: Two patients were born from the consanguineous parents. The age of onset wasabout20to25years old. Electroencephalogram showed diffuse slow waves. Brain MRIdemonstrated diffuse leukoencephalopathy and multiple subcortical infarcts. Skin and suralnerve biopsy showed concentric thickening of vascular wall, narrowing of the lumen and mildfibrous proliferation of the intima. There were no amyloid, PAS granular deposition andultrastructural granular osmiophilic materials (GOMs) on the vascular wall. No mutationswere found in exons1-33of NOTCH3gene. we identified a homozygous T to C missensemutation (c.1091T>C) in exon6in the HTRA1gene of the two patients. Both parents and theproband’s daughter had the heterozygous c.1091T>C mutation. We included100healthycontrols admitted to the sequencing of the corresponding exon and didn’t find the samemutation.Conclusion: we analyzed the first CARASIL family genetically, and found a new mutation ofHTRA1gene. Part Ⅱ Model of vascular smooth muscle cells transfected witheukaryotic expression vector of HTRA1geneObjective: To build vector of wild HTRA1gene with pEGFP-N1plasmid and mutantHTRA1gene (1091T>C) with pEGFP-N1plasmid. Then the two plasmid vectors weretransfected to vascular smooth muscle cell.Methods: Design one pairs of primer according to the CDS of HTRA1gene, the forwardprimer is5’–CTCAAGCTTCG AATTCATGCAGATCCCGCGCGCCGCTCTTC-3’, the reverse primer is5’-GGCGACCGGTGGATCC CG TGGG TCAATTTCTTCGGGAA-3’.Target gene was amplification through PCR. Join the PCR product with the p-EGFP-N1vector, then transform to E.coli and purified the plasmids. Verified by double-enzyme leavageand gene sequencing. This is wild plasmid vector(WT). In another experimental group, wedesign two primers containing the new mutation site (1091T>C) that we have already foundand use the site-directed mutagenesis method to ampliate two pieces of PCR productscontaining the new mutation site, then join the PCR products with the p-EGFP-N1vector andthe mutant plasmid vector (L364P) is obtained. Transform to E.coli and purified the plasmids.Verified by double-enzyme cleavage and gene sequencing. Human vascular smooth musclecells were cultured with SMCM. The cells were detected by immunocytochemical stainingwith α-SM-actin antibody. VSMC were divided into three groups and transiently transfectedwith plasmid by means of DNAfect.(1) WT group: transfected with wild HTRA1-pEGFP-N1plasmid.(2) L364P group: transfected with mutant HTRA1-pEGFP-N1plasmid.（3）controlgroup: transfected with pEGFP-N1plasmid. Estimate the transfection efficiency by flowcytometry.Results:(1)The OD of the wild and mutant plasmid is between1.8to2.0, the concentrition isbetween300to400ng/ul. Two products of4.7kb and1400bp were observed after doubledouble-enzyme cleavage which were corresponding with the length of pEGFP-N1vector andHTRA1gene, and were verified by sequencing.(2) The cultured cells appeared fusiform,triangle and star shapes. The characteristic peak and valley features were evident, and theywere positive to α-SM-actin antibody which indicated the cells were smooth muscle cells.The green fluorescence were observed in three group cells in24to48hours after transfection.The transfection efficiency was about33%.Conclusions: We successfully built the wild and mutant HTRA1gene plasmid vector andtransfected the vector into vascular smooth muscle cells by transient transfection method.Model of vascular smooth muscle cell with HTRA1gene was built. Part Ⅲ Expression of HTRA1gene in vascular smooth musclecellsObjective: To test the HTRA1mRNA and HtrA1expression after the wild and mutantHTRA1gene transfected into vascular smooth muscle cells. Methods: Collected the cells of three groups48hours after transfection and extrated the totalRNA and proteins. The expression of HTRA1gene was detected through real-time PCR andWestern blot method.Results: The HTRA1gene expression can be detected in the three groups. Data of RT-PCRshowed HTRA1mRNA level in L364P group and WT group were higher than that in thecontrol group, and HTRA1mRNA in L364P was lower than that in WT group. Results ofWestern blot demonstrated that the HtrA1protein expression of the two transfection groupswere high than that of the control group and the expression was less in L364group than thatin WT group.Conclusions: HTRA1gene was successfully expressed in vascular smooth muscle cells aftertransfection. The mutant HTRA1gene (1091T>C) result in the low expression of HTRA1mRNA and HtrA1protein. Part Ⅳ Fluence on TGF-β1/Smads signaling of HTRA1gene invascular smooth muscle cellsObjective: To test the change of TGF-β1/Smads signaling after the wild and mutant HTRA1gene transfected into vascular smooth muscle cells.Methods: Collected the total RNA and protein of three groups (control group, WT group andL364P group)48hours after transfection and extrated the total RNA and proteins. TGF-β1、Smad2/3/4and phosphorylated Smad2/3/were detected through real-time PCR and Westernblot method.Results: Data of RT-PCR showed mRNA level of TGF-β1and Smad2/3were higher inL364P group than that in WT group, and the differences were significant(P<0.05). The smad4mRNA level was not different in the two groups. Results of Western blot demonstrated thatthe Smad2/3protein expression in L364P group were high than that in WT group andphosphorylated Smad2/3/were detected only in L364P group. Smad4of L364P decreased alittle and the different was not significant.Conclusion: The mutant HTRA1gene (1091T>C) might increase the TGF-β1/Smadssignaling.更多还原