基质金属蛋白酶3高表达在脑动静脉畸形发生发展过程中作用机制的研究

发布时间：2018-02-26 11:21

本文关键词： 基质金属蛋白酶3 高表达脑动静脉畸形人脑微血管内皮细胞脑静脉高压大鼠模型星形胶质细胞　出处：《复旦大学》2014年博士论文　论文类型：学位论文

【摘要】：[目的]研究基质金属蛋白酶-3(MMP-3)在人脑动静脉畸形(BAVM)组织中的表达情况,通过体外实验探讨MMP-3对人脑微血管内皮细胞(HBMEC)的增殖、迁移、侵袭及血管成形能力的影响,最后通过体内实验进一步研究MMP-3表达量对脑血管结构造成的影响,从而探讨MMP-3在BAVM发生发展过程中的可能作用机制。[方法]MMPs的一些成员在BAVM组织中有异常改变,并与BAVM的发生发展有关。MMP-3是MMPs家族中的重要成员,在介导和调节其他MMPs生成及作用发挥方面具有十分重要的作用。本课题首先通过免疫组织化学及Western-blot的方法检测MMP-3在人BAVM组织中的表达情况。在此基础上通过体外实验,即构建MMP-3高表达和干扰质粒,通过质粒共转染的方法筛选出MMP-3稳定表达的HBMEC株,再通过CCK8实验、划痕实验及Transwell实验检测高表达和干扰MMP-3基因对HBMEC增殖、迁移和侵袭能力的影响。之后通过体外构建matrigel三维血管形成模型,观察高表达和干扰MMP-3基因对HBMEC血管形成能力的影响。最后,通过体内实验,即首先建立脑内静脉高压大鼠模型,在此基础上通过立体定向微量注射转染了MMP-3基因慢病毒载体的HBMEC的方法,人为造成大鼠脑内局部MMP-3表达量的变化,最终通过血管内注射荧光染料和免疫组化的方法研究MMP-3表达量对大鼠脑血管结构造成的影响。[结果]免疫组织化学及Western-blot的检测结果发现,在人BAVM组织中,MMP-3高表达于血管内皮细胞及基底膜,而正常对照组无明显表达。RT-PCR技术进一步证实,在人BAVM组织中,MMP-3基因表达量明显增加。在体外实验部分,通过质粒共转染的方法成功构建了MMP-3高表达和干扰质粒,并筛选出了稳定表达的HBMEC细胞株。CCK8实验、划痕实验及Transwell的实验结果说明MMP-3高表达可增强HBMEC的增殖、迁移和侵袭能力,而matrigel三维血管成形实验说明MMP-3基因对HBMEC的血管形成能力有重要影响。在体内实验部分,通过立体定向微量注射转染了MMP-3基因慢病毒载体的HBMEC的方法,成功建立了脑静脉高压-矢状窦旁脑组织局部MMP-3高表达大鼠模型。对该模型进行的脑血管免疫荧光实验发现,MMP-3高表达可以引起脑静脉高压-矢状窦旁脑组织局部MMP-3高表达模型大鼠的脑血管通透性的增加,即脑血管结构和功能的改变。与此同时,MMP-3高表达可以引起脑静脉高压大鼠脑微血管周围的星形胶质细胞特异性地摄取微血管内物质。[结论]本研究首次发现MMP-3高表达于人BAVM组织中,结合前期的研究结果证实MMP-3在BAVM发生发展过程中起重要作用。其作用机制可能是,一方面是通过直接和(或)影响影响HBMEC的增殖、迁移、侵袭和血管成形能力而影响脑血管新生过程,另一方面是通过诱发星形胶质细胞与脑血管内容物之间的物质交换进而改变脑血管的形态和功能。
[Abstract]:[objective] to study the expression of matrix metalloproteinase-3 (MMP-3) in human arteriovenous malformation (AVM), and to investigate the effects of MMP-3 on the proliferation, migration, invasion and angioformability of human microvascular endothelial cells (HBeMECs) in vitro. Finally, the effect of MMP-3 expression on cerebrovascular structure was studied in vivo, and the possible mechanism of MMP-3 in the pathogenesis and development of BAVM was discussed. [methods] some members of MMPs had abnormal changes in BAVM tissues. MMP-3 is an important member of the MMPs family related to the occurrence and development of BAVM. It plays a very important role in mediating and regulating the formation and function of other MMPs. Firstly, the expression of MMP-3 in human BAVM was detected by immunohistochemistry and Western-blot. The high expression and interference plasmids of MMP-3 were constructed, and the stable expression HBMEC strains of MMP-3 were screened by co-transfection. Then, the high expression and interference of MMP-3 gene on HBMEC proliferation were detected by CCK8 experiment, scratch test and Transwell experiment. The effects of high expression and interference of MMP-3 gene on the ability of HBMEC angiogenesis were observed by establishing a three-dimensional angiogenesis model of matrigel in vitro. That is to say, the rat model of cerebral venous hypertension was established at first. On the basis of this, the HBMEC of MMP-3 gene lentivirus vector was injected into the rat by stereotactic microinjection, which caused the changes of local MMP-3 expression in rat brain. Finally, the effects of MMP-3 expression on cerebral vascular structure in rats were studied by intravascular injection of fluorescent dye and immunohistochemistry. [results] Immunohistochemistry and Western-blot detection showed that, The expression of MMP-3 in human BAVM tissue was highly expressed in vascular endothelial cells and basement membrane, but not in normal control group. RT-PCR further confirmed that the expression of MMP-3 gene in human BAVM tissue was significantly increased. In the experimental part in vitro, the expression of MMP-3 gene was significantly increased. The high expression and interference plasmids of MMP-3 were successfully constructed by plasmid cotransfection, and the stable expression of HBMEC cell line. CCK8 was screened out. The results of scratch test and Transwell showed that the high expression of MMP-3 could enhance the proliferation, migration and invasion of HBMEC. Matrigel three-dimensional angioplasty experiment showed that MMP-3 gene had an important effect on the angiogenesis ability of HBMEC. In vivo, the HBMEC vector of MMP-3 gene lentivirus was transfected by stereotactic microinjection. A rat model of high expression of MMP-3 in cerebral venous hypertension and paragittal sinusoidal tissue was successfully established. The results of cerebral vascular immunofluorescence assay showed that the high expression of MMP-3 could induce cerebral venous hypertension and local brain tissue around sagittal sinus. The increase of cerebral vascular permeability in rats with high expression of MMP-3, At the same time, the high expression of MMP-3 could induce the specific uptake of microvascular substances by astrocytes around the cerebral microvascular in rats with cerebral venous hypertension. [conclusion] the high MMP-3 level was first found in this study. Expressed in human BAVM, In combination with previous studies, it is confirmed that MMP-3 plays an important role in the development of BAVM. The mechanism may be that, on the one hand, it affects the proliferation and migration of HBMEC through direct and / or influence. Invasiveness and angioformability affect the process of cerebral angiogenesis. On the other hand, it changes the morphology and function of cerebral vessels by inducing the exchange of substances between astrocytes and cerebrovascular contents.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R743.4

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