新生隐球菌通过S100A10影响MMP9侵袭血脑屏障的机制研究

发布时间：2018-06-08 17:00

本文选题：新生隐球菌 + 慢病毒　；参考：《第二军医大学》2017年硕士论文

【摘要】：研究背景隐球菌(Cryptococcus)系环境腐生菌,也是重要的机会致病真菌,常在免疫功能低下人群引起感染。中枢神经系统感染是隐球菌病最主要的临床表现,其中隐球菌性脑膜炎/脑膜脑炎(Cryptococca meningitis/meningoencephalitis,CME)是其高病死率的最主要病因。强烈的嗜中枢性是隐球菌病的一个显著特点,有超过90%的隐球菌病侵犯中枢神经系统,但目前其机制不明。血脑屏障便是隐球菌侵袭中枢神经系统的必经关卡,所以新生隐球菌侵袭血脑屏障(Blood-Brain Barrier,BBB)的途径只能是:或穿过脑微血管内皮细胞;或穿越细胞间的紧密连接结构。目前有三个机制假说:1.细胞旁途径;2.“特洛伊木马”机制;3.跨细胞途径(即内皮细胞胞吞胞吐)。S100A10蛋白是钙结合蛋白家族中S100蛋白家族中的一员,存在于很多细胞的胞浆和胞核中。S100A10蛋白可与膜联蛋白A2(annexin A2)结合为异四聚体(S100A10)2-(annexinA2)2并作为膜蛋白存在于多种细胞的细胞膜上。继而使纤溶酶原被tPA(组织型纤溶酶原激活剂)、uPA(尿激酶型纤溶酶原激活剂)激活为纤溶酶,同时激活基质金属蛋白酶,降解细胞基质。它与细胞的胞吞胞吐、物质转运等密切相关。MMP9蛋白是一类结构高度同源的分泌型或膜相关性锌内肽酶MMPs(Matrix metalloproteinase,MMP)家族中的一员,包括分子量为92kDa的无活性形式ProMMP9和82kDa的活性形式MMP9。体内绝大多数细胞并不储备MMPs,只有当需要MMPs的信号传递到细胞后才临时合成,然后以无活性的酶原形式分泌到胞外。随后被激活并呈现瀑布效应。MMPs与血脑屏障开放有关,其中MMP-9可分解IV、V、VII、X、XI、弹性蛋白、微纤维蛋白、层粘连蛋白、骨连蛋白。针对隐球菌感染血脑屏障内皮细胞后上调S100A10是否影响MMP9的表达,本研究进行了深入研究,进一步验证MMP9是否在新生隐球菌的嗜中枢性机制中起到一定作用,并探索抑制MMP9来保护血脑屏障的新方法。研究目的第一,观察新生隐球菌通过小鼠脑微血管内皮细胞(b.End3)S100A10影响基质金属蛋白酶9(MMP9)在mRNA水平和细胞内外蛋白质水平的表达;第二,沉默小鼠脑微血管内皮细胞MMP9基因后,验证基质金属蛋白酶9表达情况;构建该细胞系体外血脑屏障模型,探究此时新生隐球菌侵袭血脑屏障的能力;第三,探索基质金属蛋白酶抑制剂GM6001对新生隐球菌穿过血脑屏障是否有一定的保护作用,为进一步临床应用研究提供体外实验基础。研究方法第一部分将慢病毒转染小鼠脑微血管内皮细胞(b.end3)细胞,分别筛选稳定下调s100a10、mmp9以及空载的细胞系,分别用荧光显微镜和qpcr定性和定量验证其下调程度(该部分已发表)。以新生隐球菌b3501感染由lv-muss100a10-shrnab.end3和ncb.end3构建的简易体外血脑屏障模型,在mrna水平和蛋白水平检测基质金属蛋白酶9(mmp9)表达量的差异。第二部分将lv-musmmp9-shrnab.end3和ncb.end3分别构建transwell体外血脑屏障模型,新生隐球菌b3501感染该模型,检测其穿透血脑屏障的活菌数和bbb的完整性。第三部分基质金属蛋白酶抑制剂在新生隐球菌性脑炎/脑膜炎防治中的作用初步探索。以小鼠脑微血管内皮细胞(b.end3)细胞构建体外血脑屏障模型,放置于不同浓度的gm6001环境中。检测gm6001作用时,穿过血脑屏障的活性隐球菌数量以及血脑屏障稳定性的改变程度,以探讨gm6001对血脑屏障的保护作用及最佳浓度。结果第一部分成功构建了ncb.end3细胞系、lv-muss100a10-shrnab.end3细胞系和lv-musmmp9-shrnab.end3细胞系,后两者mmp9基因表达下调分别在76%和78.8%。经s-n-k检验,lv-muss100a10-shrnab.end3细胞系和lv-musmmp9-shrnab.end3细胞系与nc组、b.end3组差别有统计学意义,p0.05,而b.end3组与nc组的mmp9表达的差异无统计学意义(该部分已发表,见附录)。ncb.end3组与lv-muss100a10-shrnab.end3设置对照实验,两组分别与新生隐球菌b3501构建相应体外血脑屏障感染模型。mrna水平和细胞内外蛋白水平的检测发现,lv-muss100a10-shrnab.end3组在s100a10下调后,基质金属蛋白酶9(mmp9)表达量与ncb.end3组mmp9表达量的差异有统计学意义(p0.05),且均较nc组低。第二部分lv-musmmp9-shrnab.end3和空载ncb.end3分别构建体外血脑屏障的新生隐球菌感染模型。验证表明,mmp9下调后,在mrna水平和细胞内外蛋白水平分别比较两组间基质金属蛋白酶9(mmp9)表达量的差异,经检验差异有统计学意义,p0.05。在此基础上,用一定量的新生隐球菌b3501感染相应分别构建好的两组transwell体外血脑屏障模型模型,检测血脑屏障稳定性的下降程度和穿透血脑屏障模型的活菌数,以证明其在不同血脑屏障模型的侵袭能力。结果发现:第一,LV-musMMP9-shRNA b.End3所构建的血脑屏障模型,其跨内皮细胞电阻(TEER)的变化幅度与NC-b.End3组相比有明显差异,且有统计学意义(P0.05)。两组BBB的TEER变化幅度有差异,而由LV-musMMP9-shRNA b.End3所构建的血脑屏障模型在经历新生隐球菌侵袭时TEER变化幅度小,稳定性更高,完整性更好。第二,体外血脑屏障模型中,NC-b.End3组与LV-musMMP9-shRNA b.End3组的transwell下室内新生隐球菌数目随时间延长而增多,且两组各时点菌落负荷均出现差异,经成组T检验,P0.05,各时点两组差异均具有统计学意义。第三部分小鼠脑微血管内皮细胞bEnd.3构建血脑屏障模型,放置于不同浓度金属蛋白酶抑制剂GM6001的微环境,统计B3501穿过血脑屏障的数目。结果提示,GM6001 10μM处理组,transwell下室的活性菌数量减少,各组间差异有统计学意义。结论MMP9在新生隐球菌通过细胞旁途径穿透血脑屏障过程中起到重要作用,其表达水平依赖于S100A10的表达情况。隐球菌嗜中枢感染时,金属蛋白酶抑制剂可作为一种血脑屏障保护剂,缓解并减少感染的扩散,为临床辅助治疗隐球菌性脑炎/脑膜炎提供一种新的可能性。
[Abstract]:Cryptococcus neoformans (Cryptococcus), an important opportunistic pathogenic fungus, is an important opportunistic pathogenic fungus and often causes infection in people with low immune function. Central nervous system infection is the most important clinical manifestation of cryptococcosis, and cryptococcal meningitis / meningoencephalitis (Cryptococca meningitis/meningoencephalitis, CME) is a high mortality. The most important cause of the rate is the strong centrism is a prominent feature of cryptococcosis, with more than 90% of cryptococcosis invading the central nervous system, but the mechanism is unclear. The blood brain barrier is a necessary barrier for Cryptococcus neoformans to attack the central nervous system, so Cryptococcus neoformans invades the blood brain barrier (Blood-Brain Barrier, BBB). Only: or through the cerebral microvascular endothelial cells; or through the close connections between cells. There are three mechanism hypotheses: 1. cell side pathways; 2. "Troy Trojan" mechanism; 3. cross cell pathway (i.e. endothelin) is a member of the S100 protein family in the family of calcium binding proteins and exists in many cells. The.S100A10 protein in the cytoplasm and nucleus can be combined with the membrane protein A2 (annexin A2) as the heterogeneous four polymer (S100A10) 2- (annexinA2) 2 and as membrane protein exists on the cell membrane of a variety of cells. Then the plasminogen activator of the fibrinolytic enzyme is activated by tPA (tissue type plasminogen activator) and uPA (urokinase type plasminogen activator) as a fibrinolytic enzyme, and activates the plasminogen activator. .MMP9 protein is a member of a family of highly homologous secretory or membrane related zinc endopeptidase MMPs (Matrix metalloproteinase, MMP), including the active form of 92kDa and the active form MMP9. of 92kDa and 82kDa. Most of the cells in the body do not reserve MMPs, and they are temporarily synthesized only when the signal of MMPs is passed to the cell, and then secreted to the extracellular in the form of inactive zymogen. Then it is activated and presents the waterfall effect.MMPs related to the opening of the blood brain barrier, in which MMP-9 can decompose IV, V, VII, X, XI, elastin, microfibrin, laminin. This study conducted in-depth studies to further verify whether MMP9 plays a role in the central mechanism of Cryptococcus neoformans, and explores a new method to inhibit the protection of the blood brain barrier by MMP9. The first of this study is to observe the new purpose of the study. Cryptococcus neoformans affects the expression of matrix metalloproteinase 9 (MMP9) at mRNA level and protein level inside and outside of the mouse brain microvascular endothelial cells (b.End3). Second, the expression of matrix metalloproteinase 9 is verified after the silence of the MMP9 gene of the mouse brain microvascular endothelial cells, and the blood brain barrier model in vitro is constructed. At this time, the ability of Cryptococcus neoformans to invade the blood brain barrier; third, to explore the protective effect of matrix metalloproteinase inhibitor GM6001 on Cryptococcus neoformans through the blood brain barrier, and to provide the experimental basis for further clinical application. The first part of the research method transfected the lentivirus into the mouse brain microvascular endothelial cells (b.end3 The cells were screened for the stable downregulation of S100A10, MMP9 and unloaded cell lines. The down-regulation of the cells was qualitatively and quantitatively verified by fluorescence microscopy and qPCR, respectively. The simple external blood brain barrier model constructed by lv-muss100a10-shrnab.end3 and ncb.end3 for Cryptococcus neoformans b3501 infection was detected at mRNA level and protein level. The difference in the expression of matrix metalloproteinase 9 (MMP9). The second part constructs a Transwell in vitro blood brain barrier model of Transwell and ncb.end3 respectively. Cryptococcus neoformans b3501 infects the model to detect the number of living bacteria that penetrate the blood brain barrier and the integrity of BBB. The third part of the matrix metalloproteinase inhibitor in Cryptococcus neoformans Preliminary exploration of the role of encephalitis / meningitis prevention and control. A mouse brain microvascular endothelial cell (b.end3) cell is used to construct an in vitro blood brain barrier model, which is placed in a different concentration of GM6001 environment. The number of active Cryptococcus through the blood brain barrier and the change of the stability of the blood brain barrier are detected by the detection of the effect of GM6001 on the blood brain screen, in order to explore the effect of GM6001 on the blood brain screen. Results the first part successfully constructed ncb.end3 cell line, lv-muss100a10-shrnab.end3 cell line and lv-musmmp9-shrnab.end3 cell line. The down regulation of MMP9 gene expression in the latter two was 76% and 78.8%. by s-n-k test, lv-muss100a10-shrnab.end3 fine cell line and lv-musmmp9-shrnab.end3 cell line and NC group, B, respectively, B. The difference in.End3 group was statistically significant, P0.05, but there was no significant difference in the expression of MMP9 in group b.end3 and NC group (this part has been published, see Appendix).Ncb.end3 group and lv-muss100a10-shrnab.end3 set a control experiment. The two groups were constructed with Cryptococcus neoformans b3501 to construct the corresponding.Mrna level and intracellular protein of the blood brain barrier infection model in vitro. It was found that the expression of matrix metalloproteinase 9 (MMP9) expression and MMP9 expression in group ncb.end3 were significantly different from that of group ncb.end3 (P0.05), and were lower than those in the NC group after the downregulation of S100A10 in the lv-muss100a10-shrnab.end3 group. The model of Cryptococcus neoformans infection in the blood brain barrier was constructed by second partial lv-musmmp9-shrnab.end3 and no load ncb.end3. The results showed that the difference in the expression of matrix metalloproteinase 9 (MMP9) between the two groups was compared between the two groups after the downregulation, and the difference was statistically significant. On the basis of p0.05., two groups of Transwell in vitro blood brain barrier model model established by a certain amount of Cryptococcus neoformans b3501 infection were constructed. Type, detection of the decline in the stability of blood brain barrier and the number of living bacteria that penetrated the blood brain barrier model to prove its invasion ability in different blood brain barrier models. The results were as follows: first, the blood brain barrier model constructed by LV-musMMP9-shRNA b.End3 was significantly different from that of the NC-b.End3 group. And there was statistical significance (P0.05). The TEER changes in the two group of BBB were different, and the blood brain barrier model constructed by LV-musMMP9-shRNA b.End3 had little change, higher stability and better integrity during the invasion of Cryptococcus neoformans. Second, in the model of blood brain barrier in vitro, NC-b.End3 group and LV-musMMP9-shRNA b.End3 group Transwell. The number of Cryptococcus neoformans increased with time, and the colony load of the two groups at each time point was different. The difference between the two groups was statistically significant after the group T test, P0.05 and each time point. The third part of the mouse brain microvascular endothelial cells (bEnd.3) constructed the blood brain barrier model, which was placed at different concentrations of the metalloproteinase inhibitor GM6001. Microenvironment, statistical B3501 passes through the number of blood brain barriers. The results suggest that the number of active bacteria in the Transwell lower chamber is reduced in the GM6001 10 M treatment group. Conclusion MMP9 plays an important role in the process of penetrating the blood brain barrier through the paracellular pathway of Cryptococcus neoformans. The expression level of Cryptococcus neoformans depends on the expression of S100A10. When Cryptococcus neoformans are central infection, the inhibitor of metalloproteinase can be used as a protective agent for blood brain barrier to alleviate and reduce the spread of infection. It provides a new possibility for clinical adjuvant treatment of cryptococcal encephalitis / meningitis.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R519

【参考文献】