黑龍江立克次體與血管內(nèi)皮細(xì)胞及樹突狀細(xì)胞相互作用的研究
發(fā)布時(shí)間：2018-08-12 20:14
【摘要】：黑龍江立克次體(Rickettsia heilongjiangensis)是在我國(guó)首先分得的斑點(diǎn)熱群立克次體新種,是遠(yuǎn)東蜱傳斑點(diǎn)熱(Far Eastern tick-borne spotted fever)的病原體。黑龍江立克次體為專性細(xì)胞內(nèi)寄生的革蘭氏陰性菌,血管內(nèi)皮細(xì)胞是其主要靶細(xì)胞,外膜蛋白B (OmpB)是黑龍江立克次體最主要的表面蛋白抗原。 我們依據(jù)文獻(xiàn)建立體外分離、原代培養(yǎng)人臍靜脈內(nèi)皮細(xì)胞(human umbilical vein endothelial cell,HUVEC)的方法,以傳代培養(yǎng)第3代的HUVEC作為黑龍江立克次體與宿主細(xì)胞相互作用的實(shí)驗(yàn)細(xì)胞。用Vero細(xì)胞培養(yǎng)并用泛影葡胺密度梯度純化的黑龍江立克次體感染HUVEC。采用間接免疫熒光檢測(cè)和掃描電鏡觀察不同時(shí)相宿主細(xì)胞內(nèi)黑龍江立克次體,發(fā)現(xiàn)黑龍江立克次體感染HUVEC的24h內(nèi),在第6h及第24h各出現(xiàn)一個(gè)感染高峰,與文獻(xiàn)報(bào)告立氏立克次體感染HUVEC的高峰出現(xiàn)時(shí)間基本一致。在黑龍江立克次體感染HUVEC的12d內(nèi),早期立克次體無(wú)顯著增殖,感染后第5～9d,立克次體在宿主細(xì)胞內(nèi)快速增殖并播散感染相鄰細(xì)胞,感染后第10～12d,宿主細(xì)胞胞質(zhì)內(nèi)彌漫生長(zhǎng)黑龍江立克次體,宿主細(xì)胞明顯病變并脫落。結(jié)果表明黑龍江立克次體能夠感染血管內(nèi)皮細(xì)胞,在血管內(nèi)皮細(xì)胞內(nèi)不斷增殖而使宿主細(xì)胞損傷和死亡。 通過(guò)計(jì)算機(jī)抗原表位預(yù)測(cè),本研究采用PCR將黑龍江立克次體外膜蛋白B基因(ompB,4 875bp)分4段擴(kuò)增,將4個(gè)ompB基因片段分別做原核表達(dá),成功制備出4個(gè)重組OmpB蛋白(OmpB-P1、OmpB-P2、OmpB-P3、OmpB-P4)。免疫印跡分析證明4個(gè)重組OmpB蛋白均能與黑龍江立克次體感染血清特異性反應(yīng)。用純化的4個(gè)重組蛋白分別免疫C3H/HeN小鼠,IFA檢測(cè)血清抗體效價(jià)表明4個(gè)OmpB蛋白均能有效地誘導(dǎo)機(jī)體產(chǎn)生特異性體液免疫應(yīng)答(抗體滴度≥5 120),顯示4個(gè)重組OmpB蛋白具有良好的免疫原性。 樹突狀細(xì)胞(dendritic cells,DC)是專職捕獲和處理抗原并將抗原提呈給淋巴細(xì)胞的免疫細(xì)胞,可使機(jī)體產(chǎn)生免疫或耐受。本研究將4個(gè)重組OmpB蛋白分別刺激體外誘導(dǎo)培養(yǎng)的小鼠骨髓源DC,24h后用流式細(xì)胞儀分析抗原刺激的DC表型,結(jié)果發(fā)現(xiàn)4個(gè)重組OmpB蛋白刺激樹突狀細(xì)胞的表型分子(CD40, CD80, CD86和MHC-II)的表達(dá)均顯著高于陰性對(duì)照,使DC成熟。將4個(gè)重組OmpB蛋白刺激DC分別經(jīng)腹腔轉(zhuǎn)移至正常C3H/HeN小鼠,14d后用黑龍江立克次體毒株攻擊DC受體小鼠。攻擊后第7d用實(shí)時(shí)熒光定量PCR檢測(cè)小鼠脾、肺、肝、腦等臟器黑龍江立克次體載量。結(jié)果顯示接受黑龍江立克次體全菌抗原、OmpB-P2、OmpB-P3和OmpB-P4激活DC的小鼠立克次體載量顯著低于非抗原刺激DC受體小鼠(陰性對(duì)照),而接受OmpB-P1或OmpB的融合蛋白(TrxA)激活DC的小鼠立克次體載量與陰性對(duì)照相比無(wú)顯著性差異。說(shuō)明OmpB-P2、OmpB-P3和OmpB-P4能夠誘導(dǎo)特異性免疫保護(hù),為保護(hù)性抗原。 將4個(gè)重組OmpB蛋白刺激的樹突狀細(xì)胞分別與磁珠分選純化的小鼠脾臟CD4~+T細(xì)胞和CD8~+T細(xì)胞在體外共培養(yǎng),結(jié)果顯示與OmpB-P2、OmpB-P3或OmpB-P4激活DC相互作用的CD4~+T細(xì)胞和CD8~+T細(xì)胞的IFN-γ表達(dá)水平顯著高于與OmpB-P1激活DC相互作用的CD4~+T細(xì)胞和CD8~+T細(xì)胞,提示樹突狀細(xì)胞介導(dǎo)的抗黑龍江立克次體的免疫保護(hù)作用與抗原激活的CD4~+T細(xì)胞和CD8~+T細(xì)胞分別向Th1細(xì)胞分化和細(xì)胞毒性T細(xì)胞(CTL)分化及其高效表達(dá)的IFN-γ密切相關(guān)。
[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.
【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R363
本文編號(hào)：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.
【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R363
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，本文編號(hào)：2180238
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