MMP-9和高温对体外BBB紧密连接蛋白claudin-1的影响

发布时间：2018-04-29 09:44

本文选题：原代培养 + 脑微血管内皮细胞　；参考：《昆明医科大学》2014年硕士论文

【摘要】：[目的]卒中的发病率逐年上升,病后的高致残率让其备受关注。在卒中的进展过程中,血脑屏障(blood brain barrier,BBB)的破坏发挥着重要作用。BBB是介于血液和脑组织之间的一个对物质有选择性通过作用的动态界面,起着维持中枢神经系统(central nervous system,CNS)内环境稳定的重要作用。临床诊疗中我们发现卒中病人常伴有发热,而发热会加重BBB的破坏,进而加重脑损伤,影响预后。然而发热加重BBB破坏的具体机制还未能完全阐明。已有研究发现基质金属蛋白酶(matrix metalloproteinases, MMPs)特别是MMP-9,可能通过破坏细胞间的紧密连接(tight junction,TJ)导致BBB破坏。我们前期动物实验也发现,发热通过增高MMP-9活性,降解TJ的重要成分claudin-1蛋白,从而导致BBB通透性升高。但是在常温情况下,MMP-9是否具有破坏BBB结构claudin-1蛋白的作用,以及发热是否加剧MMP-9对claudin-1蛋白的破坏,目前仍不完全清楚。为此,本实验采用原代培养的SD大鼠脑微血管内皮细胞和星形胶质细胞通过Transwell小室共培养,构建体外BBB模型,予以外源性添加MMP-9及发热处置,并观察BBB模型紧密连接蛋白claudin-1的变化,从而探讨MMP-9及发热对体外BBB模型紧密连接蛋白claudin-1的影响。 [方法]首先用酶消化法进行SD大鼠脑微血管内皮细胞和星形胶质细胞原代及传代培养,并通过HE染色、免疫荧光法鉴定所培养的细胞为所需要的脑微血管内皮细胞和星形胶质细胞；再将2种经过鉴定传至3代的细胞通过Transwell小室进行共培养,建立体外BBB模型；并通过渗漏实验和Lucifer yellow (LY)渗透性测定,以确定成功建立了体外BBB模型；将模型随机分为37℃常温、39℃高温、37℃常温+MMP-9、39℃高温+MMP-9四组,并在30min、60min、90min进行LY渗透性测定,观察各组体外BBB模型通透性的变化；进而采用免疫组织化学法和蛋白印迹法观察各组BBB紧密连接蛋白claudin-1的变化。 [结果]1、经HE染色、免疫荧光法鉴定所培养的细胞为SD大鼠脑微血管内皮细胞和星形胶质细胞。通过Transwell小室建立的非接触BBB模型。渗漏试验结果成阳性。模型具有低通透系数(permeability coefficient,Pe,1.08±0.16x10"3cm/min)。 2、不同时间点组内比较,模型通透性实验显示,37℃常温组和37℃常温+MMP-9组,体外BBB模型的通透性无明显改变,差异无统计学意义(P0.05)。39℃高温组和39℃+MMP-9发热组,体外BBB模型的通透性随时间延长而逐渐增高,差异有统计学意义(P0.05)。 3、相同时间点组间比较,模型通透性实验显示,37℃常温+MMP-9组与37℃常温组比较,体外BBB模型的通透性无明显改变,差异无统计学意义(P0.05)。而39℃高温组与37℃常温组比较、39℃高温+MMP-9组与39℃高温组、39℃高.温+MMP-9组与37℃常温+MMP-9组比较,体外BBB模型的通透性均明显升高,差异有统计学意义(P0.05)。 4、不同时间点组内比较,免疫组织化学法显示,四组的claudin-1阳性表达细胞数差异均无统计学意义(P0.05)。而western blot法则显示,37℃常温组与37℃常温+MMP-9组的组内claudin-1阳性表达细胞数差异也无统计学意义(P0.05),但是39℃高温组和39℃高温+MMP-9组,claudin-1阳性表达细胞数随时间延长而逐渐下降,差异有统计学意义(P0.05)。 5、相同时间点组间比较,免疫组织化学法及western blot法都显示,37℃常温+MMP-9组与37℃常温组比较,紧密连接蛋白claudin-1的表达均无明显改变,差异无统计学意义(P0.05)。而39℃高温组与37℃常温组比较、39℃高温+MMP-9组与37℃常温+MMP-9组比较,紧密连接蛋白claudin-1的表达均明显下降,差异有统计学意义(P0.05)。 6、相同时间点组间比较,免疫组织化学法还显示,39℃高温+MMP-9组与39℃高温组相比,仅在90min时,claudin-1阳性表达细胞数进一步下降,差异有统计学意义(P0.05)。 7、相同时间点组间比较,Western Blot法还显示,39℃高温+MMP-9组与39℃高温组相比,claudin-1的表达均进一步下降,差异有统计学意义(P0.05)。 [结论]1.原代培养出了SD大鼠脑微血管内皮细胞和星形胶质细胞,并成功建立了体外BBB模型。 2、本实验条件下,单纯外源性添加MMP-9不能改变体外BBB模型的通透性,也不能破坏紧密连接蛋白claudin-1。 3、39℃高温能明显增加体外BBB模型的通透性,外源性添加MMP-9能进一步增加发热对体外BBB模型完整性的破坏。 4、39℃高温会导致体外BBB模型紧密连接蛋白claudin-1的表达下降；外源性添加MMP-9能进一步加重发热对体外BBB模型紧密连接蛋白claudin-1的破坏。
[Abstract]:[Objective] the incidence of stroke is increasing year by year, and the high disability rate after the disease has attracted much attention. In the process of stroke, the destruction of the blood brain barrier (BBB) plays an important role in.BBB, a dynamic interface between the blood and brain tissue, which has a selective interaction between the substance and the brain tissue, to maintain the central nervous system. (central nervous system, CNS) the important role of environmental stability in the clinical diagnosis and treatment of stroke patients often accompanied by fever, and fever will aggravate the destruction of BBB, aggravate brain damage and affect the prognosis. However, the specific mechanism of the exacerbation of BBB has not been fully elucidated. The study of matrix metalloproteinases (matrix metallop) has been found. Roteinases, MMPs), especially MMP-9, may cause BBB destruction by destroying the close connections between cells (tight junction, TJ). In our previous animal experiments, we also found that fever can degrade claudin-1 protein, an important component of TJ, by increasing the activity of MMP-9, which leads to the increase of BBB permeability. The effect of structure of claudin-1 protein and whether fever aggravates the destruction of MMP-9 to claudin-1 protein is still not completely clear. To this end, the primary cultured SD rat brain microvascular endothelial cells and astrocytes were co cultured in the Transwell chamber, and the BBB model was constructed in vitro, and the exogenous MMP-9 and fever treatment were added. The changes of tight junction protein Claudin-1 in BBB model were observed, and the effects of MMP-9 and fever on tight junction protein Claudin-1 in BBB model were explored.
[Methods] the original and passages of SD rat brain microvascular endothelial cells and astrocytes were cultured with enzyme digestion method, and HE staining was used to identify the cultured cells as required brain microvascular endothelial cells and astrocytes by immunofluorescence, and then 2 kinds of cells which were identified and passed to 3 generations were carried out through the Transwell chamber. Co culture, established in vitro BBB model, and through the leakage test and Lucifer yellow (LY) permeability determination to determine the successful establishment of the BBB model in vitro. The model was randomly divided into 37 C room temperature, 39 C high temperature, 37 C at normal temperature +MMP-9,39 temperature +MMP-9 four groups, and 30min, 60min, 90min LY permeability determination, observe each group of BBB model in vitro Immunohistochemical staining and Western blotting were used to observe the changes of BBB tight junction protein claudin-1.
[results]1, HE staining, immunofluorescence assay showed that the cultured cells were SD rat brain microvascular endothelial cells and astrocytes. The non contact BBB model established by the Transwell chamber. The results of the leakage test were positive. The model had low permeability coefficient (permeability coefficient, Pe, 1.08 + 0.16x10 "3cm/min).
2, compared with the time point group, the model permeability test showed that the permeability of BBB model in the normal temperature group and +MMP-9 group at 37 C was not significantly changed, the difference was not statistically significant (P0.05).39 C high temperature group and 39 C +MMP-9 fever group, the permeability of BBB model in vitro increased gradually with time, the difference was statistically significant (P0.05).
3, compared with the same time point group, the model permeability test showed that the permeability of BBB model in +MMP-9 group at 37 C and 37 C at normal temperature had no significant change, and the difference was not statistically significant (P0.05). The 39 C high temperature group was compared with the normal temperature group at 37 degrees C, the high temperature +MMP-9 group and 39 C high temperature group at 39 C, the high temperature group, and the temperature +MMP-9 group and 37 temperature +MMP-9 at normal temperature. Compared with the control group, the permeability of BBB model in vitro increased significantly, and the difference was statistically significant (P0.05).
4, compared with the different time point group, the immuno histochemical method showed that there was no significant difference in the number of claudin-1 positive cells in the four groups (P0.05), but the Western blot rule showed that there was no significant difference in the number of claudin-1 positive cells in the group of 37 C and 37 C at normal temperature +MMP-9 group, but there was no statistical significance (P0.05), but at 39 centigrade and 39 C In high temperature group +MMP-9, the number of claudin-1 positive cells decreased gradually with time, and the difference was statistically significant (P0.05).
5, compared with the same time point group, immunohistochemistry and Western blot method showed that there was no significant change in the expression of close connexin claudin-1 at normal temperature at 37 C and 37 C at normal temperature group, and the difference was not statistically significant (P0.05). The 39 C high temperature group was compared with the normal temperature group at 37, and the ratio of the high temperature +MMP-9 group to the 37 C at normal temperature +MMP-9 group was compared with that of the normal temperature group at 37. The expression of tight junction protein claudin-1 was significantly lower than that of the control group (P0.05).
6, compared with the same time point group, immuno histochemical method also showed that the number of claudin-1 positive expression cells in the high temperature +MMP-9 group at 39 C was further lower than that of the high temperature group at the temperature of 39 (P0.05), and the difference was statistically significant (P0.05).
7, compared with the same time point group, the Western Blot method also showed that the expression of Claudin-1 in the high temperature +MMP-9 group at 39 C was further decreased compared with the 39 C high temperature group, and the difference was statistically significant (P0.05).
[conclusion]1. primarily cultured SD rat brain microvascular endothelial cells and astrocytes, and successfully established BBB model in vitro.
2, under the experimental conditions, simply exogenous MMP-9 addition can not change the permeability of BBB model in vitro, nor destroy the tight junction protein claudin-1..
3,39 high temperature significantly increased the permeability of BBB model in vitro. Exogenous addition of MMP-9 could further increase the damage of fever to BBB model integrity in vitro.
High temperature at 4,39 C can lead to the decrease of the expression of tightly connexin Claudin-1 in the BBB model in vitro, and exogenous MMP-9 can further aggravate the destruction of the close connexin claudin-1 of the BBB model in vitro by the fever.

【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743.3

【参考文献】