【作者】 孟艳芬；

【导师】 温博海；

【作者基本信息】 中国人民解放军军事医学科学院 ， 微生物学， 2011， 博士

【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。更多还原

【Abstract】 Rickettsia heilongjiangensis is an obligate, intracellular, gram-negative bacterium that causes Far Eastern tick-borne spotted fever. It is a new species of spotted fever group rickettsia firstly isolated from Dermacentor silvarum ticks collected in Heilongjiang Province of China. Vascular endothelial cells are the main target cells of R. heilongjiangensis in infection and outer membrane protein B (OmpB) is it’s abundant surface-exposed protein.Human umbilical vein endothelial cells (HUVEC) were isolated and cultured in vitro. R. heilongjiangensis organisms cultured in Vero cells and purified by renografin gradient centrifugation were applied to infect HUVEC and rickettsiae in HUVEC were examined by both indirect immune fluorescence assay (IFA) and scanning electron micrograph (SEM) after infection. During the first 24 hours of infection, two infection peaks were found at 6 and 24 hours post-infection (pi), respectively, which is similar to R. rickettaii infection of HUVEC. Multiplication of rickettsiae and pathological changes in the host cells were observed at 5 days pi, while the amount of intracellular rickettsiae was found to markedly increase in the host cells and rickettsial dissemination between neighbor cells was observed within 5 to 9 days pi. A few of rickettsiae were observed in the nucleus of host cells that appeared severe pathological changes within 8 to 9 days pi. Due to the overwhelming rickettsial infection, most of the host cells shed 12 days pi. Our results demonstrated that R. heilongjiangensis has a capability to infect vascular endothelial cells and cause vascular injury.Outer membrane protein B gene (ompB, 4 875bp) of R. heilongjiangensis was divided into 4 fragments based on immunogenic epitope prediction of OmpB by computer analysis. Four fragments of ompB were amplified from the genomic DNA of R. heilongjiangensis, and subsequently 4 recombinant OmpB proteins (OmpB-P1, OmpB-P2, OmpB-P3, and OmpB-P4) were highly expressed in Escherichia coli cells transformed by ompB-p1-, ompB-p2-, ompB-p3-, ompB-p4-insered plasmids (pET32a), respectively. In western blotting analysis, all of the 4 recombinant OmpB proteins reacted with sera from mice infected with R. heilongjiangensis. The 4 OmpB proteins were used to immune C3H/HeN mice, respectively, and high levels of specific antibody titers (≧1 :5 120) were determined in mouse sera by IFA. The results suggest the OmpB proteins have good immunogenicity.Dendritic cells (DC) are antigen presenting cells that are specialized to capture, process, and present antigens to T lymphocytes to cause immunity or tolerance to the antigens. To investigate their role in Far Eastern tick-borne spotted fever, we analyzed the responses of murine bone marrow–derived dendritic cells (BMDCs) stimulated by whole cell antigen of R. heilongjiangensis and the 4 OmpB proteins in vitro and their protective roles of the antigen-pulsed BMDCs were evaluated. BMDCs pulsed by whole cell antigen, OmpB-P1, OmpB-P2, OmpB-P3, and OmpB-P4 were intraperitoneally transferred to C3H/HeN mice, respectively. On day 14 post-transfer of BMDCs, mice were intraperitoneally challenged with R. heilongjiangensis, and the challenged mice were sacrificed and their spleen, lung, liver, and brain were harvested on day 7 pi for detection of R. heilongjiangensis load by a quantitative PCR analysis. Compared with mice receiving unpulsed BMDCs (negative control), mice receiving BMDCs pulsed with whole cell antigen, OmpB-P2, OmpB-P3, or OmpB-P4 exhibited significantly lower R. heilongjiangensis load, while mice receiving BMDCs pulsed with OmpB-P1 or TrxA encoded by pET32a displayed high levels of R. heilongjiangensis load similar to that of negative control. This result suggests that OmpB-P2, OmpB-P3, and OmpB-P4 are protective antigens, but OmpB-P1 is not.CD4~+ and CD8~+ T cells from spleens of C3H/HeN mice immunized with antigen-pulsed BMDCs were isolated with T Cell Isolation Kit, respectively. The antigens-pulsed BMDCs were cocultured with the CD4~+ and CD8~+ T cells. Phenotypic molecules and intra-cellular cytokines of CD4~+ and CD8~+ T cells were analyzed on a FACScalibur flow cytometer. CD4~+/CD8~+ T cells from mice immunized with whole cell antigen-, OmpB-P2-, OmpB-P3-, or OmpB-P4-pulsed BMDCs were efficiently activated with high expression of IFN-γthat were significantly higher than that from mice immunized with OmpB-P1-pulsed BMDCs. This result suggests that protection against R. heilongjiangensis offered by BMDCs activated with the protective antigens were associated with proliferation and activation of Th1-type CD4~+ T cells and of Cytotoxic T lymphocyte (CTL) that are more potent to produce IFN-γagainst this intracellular bacterium.更多还原