MMP-2及发热对体外血脑屏障紧密连接蛋白occludin的影响

发布时间：2018-04-12 06:15

本文选题：血脑屏障模型 + 脑微血管内皮细胞　；参考：《昆明医科大学》2015年硕士论文

【摘要】：[目的]卒中是全球人口死亡和残疾的主要原因,其发病机制十分复杂。缺血性卒中是最常见的卒中类型,约占全部卒中的87%。血脑屏障(blood-brain barrier, BBB)是位于血液与脑组织之间的一个选择性通透的屏障体系,严格控制血管两侧的物质转运,从而维持中枢神经系统(central nervous system, CNS)内环境的相对稳定。卒中后BBB破坏直接影响CNS功能,导致血管源性脑水肿、出血性转化和神经元凋亡,加重神经功能缺损。基质金属蛋白酶(matrix metalloproteinases,MMPs)的激活被认为是破坏BBB结构和功能的重要分子机制。脑缺血时,基质金属蛋白酶-2(matrix metal loproteinase-2, MMP-2)可能通过降解内皮细胞紧密连接蛋白和基底膜成分使血管壁完整性破坏。发热是卒中患者早期常见并发症,有研究发现发热可加重卒中患者预后,而具体机制还未阐明。因此,研究发热加重卒中后脑损伤的机制,以及发热是否上调MMP-2表达而加剧对BBB紧密连接蛋白的损伤,就具有一定现实意义。为此,本课题通过原代培养SD大鼠乳鼠脑微血管内皮细胞和星形胶质细胞,建立简便、易行的能够模拟在体BBB的体外细胞模型。予以外源性添加MMP-2和发热处置干预,分析BBB模型紧密连接蛋白occludin的变化,从而探索MMP-2及发热在脑缺血后BBB破坏方面可能的作用机制,为临床缺血性卒中患者并发症的防治提供理论依据。[方法]以SD大鼠乳鼠为实验材料,采用酶消化法原代分离、纯化、传代培养获得脑微血管内皮细胞和星形胶质细胞,倒置显微镜下观察细胞的形态。经过HE染色,免疫荧光鉴定细胞及其纯度,使用培养3代的两种细胞通过Transwell小室进行共培养,构建体外BBB接触模型。并通过渗漏试验和LY渗透性测定,鉴定模型屏障功能。将构建成功的模型随机分为37℃常温、39℃发热、37℃常温+MMP-2、39℃发热+MMP-2四组,进而采用免疫细胞化学法和western blot方法观察各组模型中BBB紧密连接蛋白occludin的变化。同时用免疫细胞化学法检测37℃常温及39℃发热条件下星形胶质细胞MMP-2的表达。[结果]1.对分离培养的脑微血管内皮细胞和星形胶质细胞进行了形态学观察,呈现出各自典型的生长特征。经HE染色和免疫荧光法鉴定,细胞纯度达95%以上,符合两种细胞共培养的实验要求。2.两种细胞通过Transwell小室进行共培养,成功建立与12孔板相匹配的BBB接触模型。选择镜下细胞完整融合的模型进行4h渗漏试验,结果成阳性。LY渗透性检测模型具有低通透系数(permeability coefficient, Pe),其值为0.95 ±0.06×10-3cmin。3.免疫细胞化学法显示,相同时间点39℃发热组星形胶质细胞MMP-2阳性表达细胞数较37℃常温组增高,差异具有统计学意义(P0.05)。其中,39℃发热组随着处置时间的延长,星形胶质细胞MMP-2阳性表达细胞数亦增加,差异具有统计学意义(P0.05)。4.免疫细胞化学法和Western blot显示,相同时间点37℃常温+MMP-2组与37℃常温组比较,内皮细胞occludin的表达在60min和90min时均下降,差异有统计学意义(P0.05)。且37℃常温+MMP-2组随着处置时间的延长,内皮细胞occludin的表达有下降趋势,差异具有统计学意义(P0.05)。5.免疫细胞化学法和Western blot显示,相同时间点39℃发热+MMP-2组与39℃发热组比较,内皮细胞occludin的表达在60min和90min时均下降,差异有统计学意义(P0.05)。且39℃发热+MMP-2组随着处置时间的延长,内皮细胞occludin的表达有下降趋势,差异具有统计学意义(P0.05)。6.免疫细胞化学法和Western blot显示,相同时间点39℃发热组内皮细胞occludin的表达较37℃常温组下降,差异有统计学意义(PO.05)。且39℃发热组随着处置时间的延长,内皮细胞occludin的表达有下降趋势,差异具有统计学意义(P0.05)。7.免疫细胞化学法和Western blot还显示,相同时间点39℃发热+MMP-2组内皮细胞occludin的表达较37℃常温+MMP-2组明显下降,差异具有统计学意义(P0.05)。[结论]1.原代分离、培养出了SD大鼠乳鼠脑微血管内皮细胞和星形胶质细胞,并成功建立了符合实验需要的体外BBB接触模型。2.体外培养的星形胶质细胞可表达MMP-2,发热可上调星形胶质细胞MMP-2的表达。3.37℃常温下,外源性添加MMP-2可降低体外BBB模型内皮细胞紧密连接蛋白occludin的表达,并随时间的延长而加剧。4.39℃发热条件下,体外BBB模型内皮细胞紧密连接蛋白occludin的表达下调,并随时间的延长而加剧。5.发热进一步加剧MMP-2对体外BBB模型内皮细胞紧密连接蛋白occludin的下调,并随时间的延长而加剧。6.发热可能通过上调MMP-2的表达从而加剧体外BBB模型的破坏。
[Abstract]:[Objective] stroke is a major cause of death and disability in the world, its pathogenesis is very complex. Ischemic stroke is the most common type of stroke, 87%. blood brain barrier total stroke (blood-brain barrier BBB) is a selective permeability between the blood and brain barrier system, strictly control the transport vessel on both sides of the material, so as to maintain the central nervous system (central nervous system, CNS) in a relatively stable environment. After stroke BBB damage directly affects the function of CNS, leading to vasogenic brain edema and hemorrhagic transformation and apoptosis of neurons, aggravated neurological deficits. Matrix metalloproteinases (matrix, metalloproteinases, MMPs) activation is considered an important molecular mechanism of damage of the structure and function of BBB. Cerebral ischemia, matrix metalloproteinase -2 (MMP-2 matrix metal loproteinase-2) by degradation of endothelial cells closely connected The protein and basement membrane components of the integrity of vessel wall damage. Fever is a common complication in patients with early stroke, studies have found that fever can worsen the prognosis of patients with stroke, but the specific mechanism is still unclear. Therefore, study on the mechanism of fever aggravate brain injury following stroke, and whether fever and increased expression of MMP-2 increased on tight junction proteins BBB damage and it has a certain practical significance. Therefore, this topic through the primary culture of neonatal rat SD rat microvascular endothelial cells and astrocytes, establish a simple, can simulate the in vitro cell model of BBB. And to add MMP-2 and exogenous fever management intervention, change BBB model analysis of tight junction protein occludin thus, to explore the mechanism of MMP-2 and fever after cerebral ischemia BBB damage may, for the prevention and treatment of complications in patients with ischemic stroke and provide a theoretical basis for the method to S. D rat as experimental material, using enzyme digestion of primary separation, purification, to obtain brain microvascular endothelial cells and astrocytes were cultured. Cell morphology was observed under inverted microscope. After HE staining, immunofluorescence and cell purity, the use of two kinds of cells were co cultured for 3 generations by Transwell by in vitro BBB contact model. And through the leakage test and LY permeability determination, identification model of barrier function. The constructed model is divided for the success of 37 degrees at room temperature, 39 degrees fever, 37 degrees of room temperature +MMP-2,39 C +MMP-2 four fever group, then observe the change of BBB of tight junction protein occludin in each group by immune cells chemical method and Western blot method. At the same time. The expression of MMP-2 of astrocytes was detected by immunocytochemistry at room temperature 37 degrees and 39 degrees fever]1. under the condition of isolated brain capillaries Skin cells and astrocytes were observed, showing their typical growth characteristics. After HE staining and immunofluorescence, cell purity was more than 95%, with total of two cell culture experiments.2. two cells were co cultured with Transwell cells to establish BBB contact model matching with 12 hole plate the choice model of cell fusion. Complete endoscopic 4H leakage test, the positive.LY detection model has low permeability permeability coefficient (permeability coefficient, Pe), and its value is 0.95 + 0.06 * 10-3cmin.3. immunocytochemical method showed that the same time point 39 degrees fever group of astrocytes MMP-2 positive cells with 37 degrees at room temperature was increased, the difference was statistically significant (P0.05). Among them, 39 degrees fever group with prolonged treatment time, astrocytes and MMP-2 positive cells also increased the number of poor. Wasstatisticallysignificant (P0.05).4. immunocytochemistry and Western blot showed that the same time point of 37 DEG C at room temperature and 37 DEG C temperature group +MMP-2 group, occludin expression of endothelial cells were decreased in 60min and 90min, the difference was statistically significant (P0.05). And 37 normal C + MMP-2 group along with the prolonging of treatment time the expression of occludin in endothelial cells was decreased, the difference was statistically significant (P0.05).5. immunocytochemistry and Western blot showed that the same time point of 39 DEG C and 39 degree fever fever +MMP-2 group group, occludin expression of endothelial cells were decreased in 60min and 90min, the difference was statistically significant (P0.05). And 39 degrees fever group +MMP-2 with prolonged treatment time, the expression of occludin in endothelial cells was decreased, the difference was statistically significant (P0.05).6. immunocytochemistry and Western blot showed that the same time point of 39 DEG C The expression of occludin in endothelial cells of the fever group compared to 37 DEG C at room temperature was decreased, the difference was statistically significant (PO.05). And 39 degrees fever group along with the prolonging of treatment time, the expression of occludin in endothelial cells was decreased, the difference was statistically significant (P0.05).7. immunocytochemistry and Western blot also showed that the expression of the same time point 39 degrees fever group +MMP-2 endothelial cell occludin than 37 C ambient temperature +MMP-2 group decreased significantly, the difference was statistically significant (P0.05). Conclusion the primary separation of]1., developed a rat brain SD rat microvascular endothelial cells and astrocytes, and successfully established in accordance with the experimental requirements of in vitro BBB contact model of.2. in vitro cultured astrocytes express MMP-2,.3.37 expression can be up-regulated at room temperature heating MMP-2 of astrocytes, exogenous addition of MMP-2 can reduce the BBB model in vitro endothelial cell tight junction protein OC The expression of cludin, and with the extension of time and increase the temperature of.4.39 DEG C heating conditions, decrease the expression of BBB in endothelial cell model in vitro of tight junction protein occludin, and with the extension of time and increase the.5. heating further down-regulation of MMP-2 on endothelial cells in vitro BBB model of tight junction protein occludin, and with the extension of time and aggravation of.6. fever by upregulating the expression of MMP-2 in vitro BBB model thereby increasing damage.

【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R743.3

【引证文献】