黑龙江立克次体与血管内皮细胞及树突状细胞相互作用的研究
发布时间：2018-08-12 20:14
【摘要】：黑龙江立克次体(Rickettsia heilongjiangensis)是在我国首先分得的斑点热群立克次体新种,是远东蜱传斑点热(Far Eastern tick-borne spotted fever)的病原体。黑龙江立克次体为专性细胞内寄生的革兰氏阴性菌,血管内皮细胞是其主要靶细胞,外膜蛋白B (OmpB)是黑龙江立克次体最主要的表面蛋白抗原。 我们依据文献建立体外分离、原代培养人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC)的方法,以传代培养第3代的HUVEC作为黑龙江立克次体与宿主细胞相互作用的实验细胞。用Vero细胞培养并用泛影葡胺密度梯度纯化的黑龙江立克次体感染HUVEC。采用间接免疫荧光检测和扫描电镜观察不同时相宿主细胞内黑龙江立克次体,发现黑龙江立克次体感染HUVEC的24h内,在第6h及第24h各出现一个感染高峰,与文献报告立氏立克次体感染HUVEC的高峰出现时间基本一致。在黑龙江立克次体感染HUVEC的12d内,早期立克次体无显著增殖,感染后第5～9d,立克次体在宿主细胞内快速增殖并播散感染相邻细胞,感染后第10～12d,宿主细胞胞质内弥漫生长黑龙江立克次体,宿主细胞明显病变并脱落。结果表明黑龙江立克次体能够感染血管内皮细胞,在血管内皮细胞内不断增殖而使宿主细胞损伤和死亡。 通过计算机抗原表位预测,本研究采用PCR将黑龙江立克次体外膜蛋白B基因(ompB,4 875bp)分4段扩增,将4个ompB基因片段分别做原核表达,成功制备出4个重组OmpB蛋白(OmpB-P1、OmpB-P2、OmpB-P3、OmpB-P4)。免疫印迹分析证明4个重组OmpB蛋白均能与黑龙江立克次体感染血清特异性反应。用纯化的4个重组蛋白分别免疫C3H/HeN小鼠,IFA检测血清抗体效价表明4个OmpB蛋白均能有效地诱导机体产生特异性体液免疫应答(抗体滴度≥5 120),显示4个重组OmpB蛋白具有良好的免疫原性。 树突状细胞(dendritic cells,DC)是专职捕获和处理抗原并将抗原提呈给淋巴细胞的免疫细胞,可使机体产生免疫或耐受。本研究将4个重组OmpB蛋白分别刺激体外诱导培养的小鼠骨髓源DC,24h后用流式细胞仪分析抗原刺激的DC表型,结果发现4个重组OmpB蛋白刺激树突状细胞的表型分子(CD40, CD80, CD86和MHC-II)的表达均显著高于阴性对照,使DC成熟。将4个重组OmpB蛋白刺激DC分别经腹腔转移至正常C3H/HeN小鼠,14d后用黑龙江立克次体毒株攻击DC受体小鼠。攻击后第7d用实时荧光定量PCR检测小鼠脾、肺、肝、脑等脏器黑龙江立克次体载量。结果显示接受黑龙江立克次体全菌抗原、OmpB-P2、OmpB-P3和OmpB-P4激活DC的小鼠立克次体载量显著低于非抗原刺激DC受体小鼠(阴性对照),而接受OmpB-P1或OmpB的融合蛋白(TrxA)激活DC的小鼠立克次体载量与阴性对照相比无显著性差异。说明OmpB-P2、OmpB-P3和OmpB-P4能够诱导特异性免疫保护,为保护性抗原。 将4个重组OmpB蛋白刺激的树突状细胞分别与磁珠分选纯化的小鼠脾脏CD4~+T细胞和CD8~+T细胞在体外共培养,结果显示与OmpB-P2、OmpB-P3或OmpB-P4激活DC相互作用的CD4~+T细胞和CD8~+T细胞的IFN-γ表达水平显著高于与OmpB-P1激活DC相互作用的CD4~+T细胞和CD8~+T细胞,提示树突状细胞介导的抗黑龙江立克次体的免疫保护作用与抗原激活的CD4~+T细胞和CD8~+T细胞分别向Th1细胞分化和细胞毒性T细胞(CTL)分化及其高效表达的IFN-γ密切相关。
[Abstract]:Rickettsia heilongjiangensis (Rickettsia heilongjiangensis) is a new species of spotted fever group rickettsia, which was first isolated in China. Rickettsia heilongjiangensis is the pathogen of Far Eastern tick-borne spotted fever. Rickettsia heilongjiangensis is a gram-negative bacteria parasitic in specific cells. Vascular endothelial cells are the main target cells and the outer membrane is the main target cells. Protein B (OmpB) is the major surface protein antigen of Rickettsia Rickettsia in Heilongjiang.
We established a method of isolation and primary culture of human umbilical vein endothelial cell (HUVEC) in vitro. The third passage of HUVEC was used as the experimental cell for the interaction between Rickettsia Heilongjiang and host cells. Rickettsia Heilongjiang was detected by indirect immunofluorescence assay and scanning electron microscopy (SEM). It was found that the infection peaks of Rickettsia Heilongjiang were observed within 24 hours of infection with HUVEC, at 6 hours and at 24 hours respectively, which were basically consistent with the peak time reported in literature. Within 12 days after infection with HUVEC, early Rickettsia did not proliferate significantly. Five to nine days after infection, Rickettsia proliferated rapidly in the host cells and spread to the adjacent cells. From 10 to 12 days after infection, Rickettsia Heilongjiang diffusely grew in the cytoplasm of the host cells, and the host cells showed obvious pathological changes and abscission. Secondary organisms can infect vascular endothelial cells and proliferate continuously in vascular endothelial cells, resulting in injury and death of host cells.
In this study, four recombinant OmpB proteins (OmpB-P1, OmpB-P2, OmpB-P3, OmpB-P4) were successfully prepared by using PCR to amplify the outer membrane protein B gene (ompB, 4875 bp) of Rickettsia Heilongjiang and prokaryotic expression of the four ompB gene fragments. Four recombinant proteins were used to immunize C3H/HeN mice with Rickettsia. IFA assay showed that all of the four OmpB proteins could effectively induce specific humoral immune responses (antibody titer < 5 120), indicating that the four recombinant OmpB proteins had good immunogenicity.
Dendritic cells (DC) are immune cells specializing in capturing and processing antigens and presenting them to lymphocytes, which can induce immunity or tolerance. In this study, four recombinant OmpB proteins were used to stimulate murine bone marrow derived DC in vitro, and the phenotype of DC stimulated by antigen was analyzed by flow cytometry 24 hours later. The expression of phenotypic molecules (CD40, CD80, CD86 and MHC-II) of dendritic cells stimulated by four recombinant OmpB proteins was significantly higher than that of the negative control, which made DC mature. DCs stimulated by four recombinant OmpB proteins were transferred into normal C3H/HeN mice by abdominal cavity, and then attacked DC receptor mice by Rickettsia Heilongjiang strain 14 days later. The results showed that the Rickettsia loads of mice receiving Rickettsia Heilongjiang antigen, OmpB-P2, OmpB-P3 and OmpB-P4 activated DC were significantly lower than those of mice receiving non-antigen-stimulated DC receptor (negative control), while those receiving OmpB-P1 or OmpB fusion protein (TrxA) activated DC were significantly lower than those of mice receiving non-antigen-stimulated DC receptor (negative control). The results showed that OmpB-P2, OmpB-P3 and OmpB-P4 could induce specific immune protection and were protective antigens.
Four recombinant OmpB protein-stimulated dendritic cells were co-cultured with mouse spleen CD4~+ T cells and CD8~+ T cells separately and purified by magnetic beads. The results showed that the IFN-gamma expression levels of CD4~+ T cells and CD8~+ T cells interacting with OmpB-P2, OmpB-P3 or OmpB-P4-activated DC were significantly higher than those interacting with OmpB-P1-activated DC. Cells and CD8~+ T cells suggest that dendritic cell mediated immune protection against Rickettsia Heilongjiang is closely related to antigen-activated CD4~+ T cells and CD8~+ T cells differentiating into Th1 cells and cytotoxic T cells (CTL) and their highly expressed IFN-gamma.
【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
本文编号：2180238
[Abstract]:Rickettsia heilongjiangensis (Rickettsia heilongjiangensis) is a new species of spotted fever group rickettsia, which was first isolated in China. Rickettsia heilongjiangensis is the pathogen of Far Eastern tick-borne spotted fever. Rickettsia heilongjiangensis is a gram-negative bacteria parasitic in specific cells. Vascular endothelial cells are the main target cells and the outer membrane is the main target cells. Protein B (OmpB) is the major surface protein antigen of Rickettsia Rickettsia in Heilongjiang.
We established a method of isolation and primary culture of human umbilical vein endothelial cell (HUVEC) in vitro. The third passage of HUVEC was used as the experimental cell for the interaction between Rickettsia Heilongjiang and host cells. Rickettsia Heilongjiang was detected by indirect immunofluorescence assay and scanning electron microscopy (SEM). It was found that the infection peaks of Rickettsia Heilongjiang were observed within 24 hours of infection with HUVEC, at 6 hours and at 24 hours respectively, which were basically consistent with the peak time reported in literature. Within 12 days after infection with HUVEC, early Rickettsia did not proliferate significantly. Five to nine days after infection, Rickettsia proliferated rapidly in the host cells and spread to the adjacent cells. From 10 to 12 days after infection, Rickettsia Heilongjiang diffusely grew in the cytoplasm of the host cells, and the host cells showed obvious pathological changes and abscission. Secondary organisms can infect vascular endothelial cells and proliferate continuously in vascular endothelial cells, resulting in injury and death of host cells.
In this study, four recombinant OmpB proteins (OmpB-P1, OmpB-P2, OmpB-P3, OmpB-P4) were successfully prepared by using PCR to amplify the outer membrane protein B gene (ompB, 4875 bp) of Rickettsia Heilongjiang and prokaryotic expression of the four ompB gene fragments. Four recombinant proteins were used to immunize C3H/HeN mice with Rickettsia. IFA assay showed that all of the four OmpB proteins could effectively induce specific humoral immune responses (antibody titer < 5 120), indicating that the four recombinant OmpB proteins had good immunogenicity.
Dendritic cells (DC) are immune cells specializing in capturing and processing antigens and presenting them to lymphocytes, which can induce immunity or tolerance. In this study, four recombinant OmpB proteins were used to stimulate murine bone marrow derived DC in vitro, and the phenotype of DC stimulated by antigen was analyzed by flow cytometry 24 hours later. The expression of phenotypic molecules (CD40, CD80, CD86 and MHC-II) of dendritic cells stimulated by four recombinant OmpB proteins was significantly higher than that of the negative control, which made DC mature. DCs stimulated by four recombinant OmpB proteins were transferred into normal C3H/HeN mice by abdominal cavity, and then attacked DC receptor mice by Rickettsia Heilongjiang strain 14 days later. The results showed that the Rickettsia loads of mice receiving Rickettsia Heilongjiang antigen, OmpB-P2, OmpB-P3 and OmpB-P4 activated DC were significantly lower than those of mice receiving non-antigen-stimulated DC receptor (negative control), while those receiving OmpB-P1 or OmpB fusion protein (TrxA) activated DC were significantly lower than those of mice receiving non-antigen-stimulated DC receptor (negative control). The results showed that OmpB-P2, OmpB-P3 and OmpB-P4 could induce specific immune protection and were protective antigens.
Four recombinant OmpB protein-stimulated dendritic cells were co-cultured with mouse spleen CD4~+ T cells and CD8~+ T cells separately and purified by magnetic beads. The results showed that the IFN-gamma expression levels of CD4~+ T cells and CD8~+ T cells interacting with OmpB-P2, OmpB-P3 or OmpB-P4-activated DC were significantly higher than those interacting with OmpB-P1-activated DC. Cells and CD8~+ T cells suggest that dendritic cell mediated immune protection against Rickettsia Heilongjiang is closely related to antigen-activated CD4~+ T cells and CD8~+ T cells differentiating into Th1 cells and cytotoxic T cells (CTL) and their highly expressed IFN-gamma.
【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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