猪链球菌与人脑微血管内皮细胞相互作用研究

发布时间：2018-05-18 20:55

本文选题：猪链球菌 + 脑微血管内皮细胞　；参考：《中国人民解放军军事医学科学院》2011年博士论文

【摘要】：猪链球菌病是重要的新发传染病,1998年和2005年先后两次在我国江苏和四川爆发流行,造成200多人感染,50多人死亡,具有较高的病死率和病残率。脑膜炎为猪链球菌感染的主要临床症状。目前,猪链球菌致脑膜炎的致病机制还不完全清楚。一般认为,猪链球菌致脑膜炎过程是多步骤的,其中关键环节是:猪链球菌如何以较低水平的菌量从粘膜上皮进入血液;猪链球菌如何实现血中存活,造成菌血症;猪链球菌如何穿过血脑屏障进入中枢神经系统。猪链球菌与脑微血管内皮细胞相互作用的过程是猪链球菌穿过血脑屏障的重要过程。目前,对猪链球菌与脑微血管内皮细胞的相互作用特征有了一定的了解,如猪链球菌能粘附猪脑微血管内皮细胞(porcine brain microvascular endothelial cells, pBMEC)和人脑微血管内皮细胞(human brain microvascular endothelial cells, hBMEC),但不能侵袭hBMEC;猪链球菌能刺激BMEC细胞分泌细胞因子等。但有关猪链球菌与BMEC细胞相互作用的细胞和分子机制还不完全清楚。因此,本研究拟从三个方面研究猪链球菌与hBMEC细胞的相互作用。即:猪链球菌如何调节自身,调控特定的毒力因子应对同hBMEC细胞的接触;猪链球菌感染对hBMEC细胞基因转录的影响以及猪溶血素对hBMEC细胞骨架的影响。第一部分:猪链球菌与hBMEC接触后,自身基因表达谱变化情况。本部分选用猪链球菌DNA芯片,研究与hBMEC细胞接触后的猪链球菌全基因表达谱的变化。研究发现:在与hBMEC接触1 h后,猪链球菌有219个基因转录发生改变(其中131个基因上调,88个基因下调),与hBMEC接触3 h后,猪链球菌有175个基因转录发生改变(其中123个基因上调,52个基因下调)。根据猪链球菌05ZY基因组的注释信息,对所有表达发生改变的基因进行COG分类。COG分类显示在编码基因翻译、氨基酸运输、能量产生、细胞周期调控、细胞壁合成及脂类运输相关的基因中,表达上调的基因数目多于表达下调的基因数目;而在编码核苷酸运输和碳水化合物运输相关的基因中,表达下调的基因数目多于表达上调的基因数目。进一步的分析发现:细胞接触后,与猪链球菌荚膜(capsule, CPS)合成有关的基因表达上调。透射电子显微镜观察发现,与细胞接触后,猪链球菌荚膜明显增厚,证明猪链球菌荚膜合成相关基因在猪链球菌与细胞接触后表达上调。除上调CPS合成相关基因外,与hBMEC接触后,猪链球菌还上调了epf、mrp、ofs等毒力基因的表达,但下调了ADS和sly等毒力基因的表达。另外,与hBMEC接触后,猪链球菌上调细胞壁合成有关的基因,细胞壁蛋白基因如sao、SSU05_0272等,菌毛蛋白基因如sfp1,sfp2等以及与LTA丙氨酰化修饰有关的基因如dltB等的表达。此外,与hBMEC细胞接触后,猪链球菌上调参与脂类合成的基因,参与基因复制、转录特别是蛋白合成的基因,以及编码ATP合成酶F1F0的基因如atpC,atpD,atpA,atpH,atpF等的表达,使得代谢活动增加。同时,猪链球菌还上调细胞分裂相关基因的表达,使得细胞周期加快。在分析芯片数据的基础上,我们研究了猪链球菌与细胞接触后荚膜增厚的分子机制。实验结果表明:细胞接触后猪链球菌荚膜的增厚,同双组分系统2148/2149和Rgg有关,而同covR无关。具体机制为:猪链球菌在与hBMEC细胞接触后,Rgg能通过某种机制感知细胞接触信号,并将其传递给2148/2149基因,后者通过某种机制上调了CPS基因的表达。尽管covR能负调节CPS基因的表达,但与细胞接触后,存在某种未知的机制阻断covR对荚膜多糖合成相关基因的调控。通过对与hBMEC接触后猪链球菌基因表达谱变化的分析,我们推测猪链球菌基因表达的改变,可能有助于猪链球菌穿过血脑屏障。首先,增厚的荚膜和丙氨酰化的LTA有助于猪链球菌抵抗血中吞噬细胞的清除和阳离子抗菌肽的杀伤。而且,荚膜自身还能以TLR-2和MyD88非依赖的方式诱发MCP-1的释放。而MCP-1能下调紧密连接蛋白如ZO-1,occludin等在BMEC细胞中的表达并改变其在细胞间的分布,破坏血脑屏障的完整性。其次,猪链球菌上调编码菌毛蛋白的基因、细胞壁蛋白的基因和细胞壁合成相关的基因的表达。这些上调表达的基因有助于增强猪链球菌与hBMEC细胞受体的识别,提高猪链球菌与hBMEC细胞的粘附,有助于猪链球菌刺激hBMEC细胞分泌细胞因子。再者,猪链球菌上调细胞周期相关基因和代谢、蛋白翻译相关基因的表达。这些上调的基因,能加快猪链球菌的分裂,有助于猪链球菌在hBMEC细胞外的增殖。第二部分:猪链球菌感染对hBMEC细胞基因表达的影响。猪链球菌野生型261菌株和2148/2149基因敲除突变体分别以1:1的感染复数感染hBMEC细胞系hCMEC/D3细胞,以未感染的细胞为阴性对照,4 h后分别提取各组hCMEC/D3细胞的总RNA,逆转录和荧光标记后,同高密度的人全基因表达谱芯片(Agilent SurePrint G3 Human GE 8×60K)杂交。芯片数据分析时,我们选择q-value(%)≤5,同时差异倍数大于1.5倍的基因作为差异表达的基因。研究发现,相比未感染组细胞,261菌株感染组细胞有2780个差异表达基因,其中446个基因上调表达,2334个基因下调表达。相比未感染组细胞,2148/2149突变体感染组细胞有2926个差异表达的基因,其中611个基因上调表达,2315个基因下调表达。而261菌株感染组和2148/2149突变体感染组的hCMEC/D3细胞表达谱之间未有差异显著的基因。进一步的分析发现:在猪链球菌261菌株和2148/2149突变体感染4 h后,hCMEC/D3细胞大量上调编码细胞因子如IL-1?,IL-6,IL-11,GM-CSF和趋化因子IL-8,MCP-1,CXCL1,CXCL2等基因的表达,上调细胞粘附蛋白如selectin E,ICAM-4等基因的表达,同时下调紧密连接相关蛋白如ZO-1,claudin 5的表达。此外,编码细胞表面抗原的基因如CD34,CD59,CD93,CD83和CD274的表达也发生改变。通过对猪链球菌感染后hCMEC/D3细胞基因表达谱数据的分析,结合目前猪链球菌与hBMEC细胞相互作用的研究进展,我们推测猪链球菌可能通过脑微血管内皮细胞间隙的方式穿过血脑屏障。首先,猪链球菌对hBMEC粘附率很低,且无侵袭能力,因此猪链球菌直接穿过hBMEC细胞进入中枢神经系统的可能性基本可以排除。其次,猪链球菌感染后,hBMEC细胞编码细胞因子、趋化因子和细胞粘附蛋白的基因表达上调。这些上调表达的分子在招募和粘附中性白细胞和单核细胞方面有重要作用,而中性白细胞和单核细胞粘附内皮细胞后,能激活内皮细胞信号通路,调节内皮细胞紧密连接的分布,增大细胞间的通透性。再者,猪链球菌感染后,hBMEC细胞下调ZO-1,Claudin 5,par-6等基因的表达,而这些蛋白在维持内皮细胞间的紧密连接中有重要作用。第三部分:猪溶血素对hBMEC细胞骨架的影响。荧光显微镜观察发现:亚裂解浓度的猪链球菌上清和SLY蛋白能重塑hBMEC的细胞骨架,形成应力纤维、丝状伪足和片状伪足。SLY蛋白对hBMEC细胞骨架的重塑表现为胆固醇依赖性。与不同浓度的胆固醇预孵育后,SLY蛋白重塑细胞骨架的活性被部分或完全抑制,而用M?CD对hBMEC细胞表面的胆固醇进行预去除,同样能抑制猪链球菌上清和SLY对细胞骨架的重塑。为了研究SLY蛋白重塑hBMEC细胞骨架的分子机制,我们检测了hBMEC细胞裂解液中GTPase激活情况,研究发现猪链球菌上清和SLY均能激活hBMEC的RhoA和Rac1,且表现为时间依赖性,10 min时激活效应达到最大,但猪链球菌上清和SLY蛋白重塑hBMEC细胞骨架时没有激活Cdc42。总之,本研究从病原体和宿主细胞的反应性两方面,研究了猪链球菌与hBMEC的相互作用。病原体方面:与hBMEC接触后,猪链球菌通过双组分系统Rgg和2148/2149上调荚膜合成相关基因的表达,增厚荚膜。此外,猪链球菌上调脂磷壁酸丙氨酰化修饰相关的基因。增厚的荚膜和丙氨酰化的LTA有助于猪链球菌抵抗吞噬细胞和阳离子抗菌肽的杀伤。另外,猪链球菌还上调表达编码菌毛蛋白的基因、细胞壁合成相关的基因以及细胞壁蛋白的基因,这有助于重塑猪链球菌的表面。猪链球菌表面蛋白表达量的上调,有助于猪链球菌与hBMEC的接触,有助于猪链球菌刺激宿主细胞分泌细胞因子并激活其信号通路。此外,猪链球菌还上调细胞分裂相关基因的表达,加快细胞周期,这些变化有助于猪链球菌在细胞表面的增殖。宿主细胞方面:猪链球菌感染后,hBMEC细胞大量上调编码细胞因子、趋化因子及细胞粘附蛋白基因的表达,下调编码紧密连接相关蛋白基因的表达,同时改变细胞表面抗原基因表达。这些变化有助于提高脑微血管内皮细胞间的通透性。此外,猪链球菌上清和猪溶血素还能通过激活RhoA和Rac1重塑hBMEC细胞的细胞骨架,形成应力纤维、丝状伪足和片状伪足。由于肌动蛋白同紧密连接分子相连,细胞骨架的重塑造成紧密连接蛋白的重新分布,增大细胞间的间隙。我们推测这些变化有利于猪链球菌从脑微血管内皮细胞间隙穿过血脑屏障。
[Abstract]:Streptococcus suis disease (Streptococcus suis) is an important new infectious disease. It broke out in Jiangsu and Sichuan two times in China in 1998 and 2005, resulting in infection of more than 200 people, more than 50 deaths, high mortality and morbidity. Meningitis is the main clinical symptom of Streptococcus suis infection. The pathogenesis of meningitis caused by Streptococcus suis is not completely clear at present. It is generally believed that the process of meningitis induced by Streptococcus suis is a multistep process, and the key link is how Streptococcus suis enters the blood from the mucosal epithelium at a lower level of bacteria, how Streptococcus suis can survive in the blood, cause bacteremia, and how Streptococcus suis enters the central nervous system through the blood brain barrier.
The interaction between Streptococcus suis and cerebral microvascular endothelial cells is an important process of Streptococcus suis passing through the blood brain barrier. At present, the interaction characteristics of Streptococcus suis and cerebral microvascular endothelial cells have some understanding, for example, Streptococcus suis can adhere to porcine brain microvascular endothelial cells. PBMEC) and human brain microvascular endothelial cells (human brain microvascular endothelial cells, hBMEC), but not hBMEC, Streptococcus suis can stimulate the secretion of cytokines in BMEC cells, but the cell and molecular mechanisms of the interaction between Streptococcus suis and BMEC cells are not completely clear.
Therefore, this study intends to study the interaction between Streptococcus suis and hBMEC cells from three aspects: how Streptococcus suis regulates itself, regulates specific virulence factors to deal with hBMEC cells, the effect of Streptococcus suis infection on the gene transcription of hBMEC cells and the effect of pig hemolysin on the cytoskeleton of hBMEC.
Part one: the change of gene expression profile in Streptococcus suis after contact with hBMEC.
In this part, Streptococcus suis DNA chip was used to study the whole gene expression profiles of Streptococcus suis after contact with hBMEC cells. It was found that after 1 h contact with hBMEC, Streptococcus suis had 219 gene transcriptional changes (131 of them up, 88 genes down). After 3 h contact with hBMEC, there were 175 gene transcriptional changes in Streptococcus suis. Change (123 of these genes up, 52 genes down). According to the annotation information of the 05ZY genome of Streptococcus suis, the COG classification of all the genes that have changed is classified by.COG classification in the encoding gene translation, amino acid transport, energy production, cell cycle regulation, cell wall synthesis, and lipid transport related genes. The number of down regulated genes was more than the number of genes expressed. In genes encoding nucleotide transport and carbohydrate transport, the number of down regulated genes was more than the number of genes up - regulated.
Further analysis showed that after cell contact, the gene expression related to the synthesis of capsule (CPS) was up-regulated. Transmission electron microscopy showed that after contact with the cells, the capsule of Streptococcus suis was thickened obviously. It was proved that the gene of Streptococcus suis was up regulated by Streptococcus suis after contact with the cells. In addition to the up regulation of CPS In contact with hBMEC, Streptococcus suis also up-regulated the expression of EPF, MRP, ofs and other virulence genes, but down regulated the expression of virulence genes such as ADS and sly. In addition, Streptococcus suis up-regulated the genes related to cell wall synthesis, cell wall protein genes such as Sao, SSU05_0272, and pilin protein genes such as sfp1, SFP2 and so on. In addition to the expression of LTA propanylated modification related genes such as dltB, in addition, after contact with hBMEC cells, Streptococcus suis up-regulated genes involved in lipid synthesis, participated in gene replication, transcriptional in particular protein synthesis genes, and the expression of the genes encoding ATP synthase F1F0, such as atpC, atpD, atpA, atpH, atpF, etc., to increase metabolic activity. Streptococcus suis also up-regulated the expression of cell division related genes and accelerated cell cycle.
On the basis of the analysis of the chip data, we studied the molecular mechanism of the capsule thickening of Streptococcus suis after contact with the cells. The experimental results showed that the thickening of the capsule of Streptococcus suis after contact was related to the two component system 2148/2149 and Rgg, but not with covR. The specific mechanism is that after contact with hBMEC cells, the Streptococcus suis can pass through some sort. The mechanism perceiving the cell contact signal and transferring it to the 2148/2149 gene, the latter up-regulated the expression of the CPS gene through some mechanism. Although covR can negatively regulate the expression of CPS gene, there is a certain unknown mechanism that blocks the regulation of covR on the related basis of the capsule polysaccharide synthesis after contact with the cells.
By analyzing the gene expression profiles of Streptococcus suis after contact with hBMEC, we speculate that the changes in gene expression of Streptococcus suis may help Streptococcus suis to pass through the blood brain barrier. First, the thickened capsule and propionylated LTA can help Streptococcus suis to resist the clearance of phagocytes in blood and the killing of cationic antimicrobial peptides. The membrane itself can also induce the release of MCP-1 in a non dependent manner of TLR-2 and MyD88. And MCP-1 can down regulate the expression of tight connexin such as ZO-1, occludin and so on in BMEC cells and change its distribution in the cell and destroy the integrity of the blood brain barrier. Secondly, Streptococcus suis is up regulation of the gene of protein hair protein, gene and fine of cell wall protein. These up-regulated genes help to enhance the recognition of Streptococcus suis and hBMEC cell receptors, increase the adhesion of Streptococcus suis to hBMEC cells and stimulate Streptococcus suis to stimulate the secretion of cytokines in hBMEC cells. Furthermore, Streptococcus suis is up regulation of cell cycle related genes and metabolism, and related to protein translation These up-regulated genes can accelerate the division of Streptococcus suis and contribute to the proliferation of Streptococcus suis in hBMEC cells.
The second part: the effect of Streptococcus suis infection on the gene expression of hBMEC cells.
Streptococcus suis wild type 261 and 2148/2149 gene knockout mutants infect hBMEC cell line hCMEC/D3 cells with 1:1 infection complex number respectively, and the uninfected cells were negative control. The total RNA of hCMEC/D3 cells in each group was extracted after 4 h. After reverse transcription and fluorescence labeling, the same high density human whole gene expression spectrum chip (Agilent SurePrint) G3 Human GE 8 x 60K) hybridization.
In the analysis of chip data, we chose Q-value (%) (%) less than 5 and 1.5 times more than 1.5 times as differentially expressed genes. It was found that there were 2780 differentially expressed genes in the infected cells of 261 strains compared to those in the uninfected group, of which 446 genes were up-regulated and 2334 genes were downregulated. Compared with the uninfected group, 2148/2149 There were 2926 differentially expressed genes in the mutant infection group, of which 611 genes were up-regulated and 2315 genes were down regulated. There was no significant difference in the hCMEC/D3 cell expression profiles between the 261 infection group and the 2148/2149 mutant infection group.
Further analysis showed that after Streptococcus suis 261 and 2148/2149 mutants were infected with 4 h, hCMEC/D3 cells increased the expression of encoding cytokines such as IL-1?, IL-6, IL-11, GM-CSF and chemokine IL-8, MCP-1, CXCL1, CXCL2 and so on, up regulation of the expression of cell adhesion proteins such as selectin, and down regulated closely connected phase In addition, the expression of genes encoding cell surface antigens, such as CD34, CD59, CD93, CD83 and CD274, also changed, such as ZO-1, claudin 5.
Through the analysis of the gene expression profiles of hCMEC/D3 cells after Streptococcus suis infection and the research progress of the interaction between Streptococcus suis and hBMEC cells, we speculate that Streptococcus suis may pass through the blood brain barrier through the intercellular space of the cerebral microvascular endothelial cells. First, the adhesion rate of Streptococcus suis to hBMEC is very low and has no invasion ability. Therefore, the possibility of Streptococcus suis directly through hBMEC cells into the central nervous system can be eliminated. Secondly, after Streptococcus suis infection, hBMEC cells encode cytokines, chemokines and cell adhesion proteins. These up-regulated molecules are heavy in the recruitment and adhesion of neutrophils and mononuclear cells. When the neutrophils and mononuclear cells adhere to the endothelial cells, it can activate the endothelial cell signaling pathway, regulate the distribution of the endothelial cells tightly connected, and increase the permeability of the cells. Furthermore, after Streptococcus suis infection, hBMEC cells downregulate the expression of ZO-1, Claudin 5, Par-6 and other basic factors, and these proteins are tight between the endothelial cells. There is an important role in dense connections.
The third part: the effect of porcine hemolysin on hBMEC cytoskeleton.
The fluorescence microscopy showed that the sublysing concentration of Streptococcus suis supernatant and SLY protein could reshape the cytoskeleton of hBMEC and form stress fibers. The remolding of hBMEC cytoskeleton by filamentous and flaky pseudo foot.SLY protein was cholesterol dependent. After incubating with different concentrations of cholesterol, the activity of the remolded cytoskeleton of the SLY protein was found. Partial or complete inhibition, the removal of cholesterol on the surface of hBMEC cells by M? CD can also inhibit the reconstitution of cytoskeleton by supernatants of Streptococcus suis and SLY.
In order to study the molecular mechanism of SLY protein remolding hBMEC cytoskeleton, we detected the activation of GTPase in hBMEC cell lysate. The study found that Streptococcus suis supernatant and SLY could activate hBMEC RhoA and Rac1, and were time dependent, and the activation effect reached the maximum at 10 min, but Streptococcus suis supernatant and SLY protein reshaped hBMEC cells. Cdc42. does not activate the skeleton
In this study, the interaction between Streptococcus suis and hBMEC was studied from two aspects of the reactivity of the pathogen and host cell. In the aspect of pathogen: Streptococcus suis is up regulation of the expression of the related genes in the capsule synthesis and the thickening capsule through the dual component system Rgg and 2148/2149. In addition, Streptococcus suis is up regulation of lipophosphoric acid propanolylation. Decorrelating genes. Thickened capsule and propionylated LTA can help Streptococcus suis to resist the killing of phagocytes and cationic antimicrobial peptides. In addition, Streptococcus suis also up-regulated genes encoding pili protein, cell wall synthesis related genes and cell wall protein genes, which helps to reshape the surface of Streptococcus suis. The up-regulated expression of the surface protein of the bacteria contributes to the contact of Streptococcus suis with hBMEC, which helps to stimulate the secretion of cytokines and activate its signaling pathway by Streptococcus suis. In addition, Streptococcus suis also up-regulated the expression of cell division related genes and accelerated the cell cycle. These changes contribute to the proliferation of Streptococcus suis on the cell surface. Main cell side: after Streptococcus suis infection, hBMEC cells up regulate the expression of encode cytokines, chemokines and cell adhesion protein genes, down regulate the expression of closely linked protein genes and change the expression of cell surface antigen gene. These changes help to improve the permeability of cerebral microvascular endothelial cells. Streptococcus suis supernatant and pig hemolysin can also remould the cytoskeleton of hBMEC cells by activating RhoA and Rac1 to form stress fibers, filamentous and flaky pseudo feet. The redistribution of close connexin resulting from the remolding of the cytoskeleton caused by actin is connected to the close connexion molecules, and the intercellular space is increased. It helps Streptococcus suis cross the blood-brain barrier from the gap of brain microvascular endothelial cells.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R363

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