【作者】 李嫄渊；

【导师】 黄瑞； 吴淑燕；

【作者基本信息】 苏州大学 ， 病原生物学， 2010， 博士

【摘要】 目的:通过细胞模型和实验动物模型,研究沙门菌毒力基因(Salmonella plasmid virulence genes,spv)对树突状细胞(dendritic cells,DCs)自噬、凋亡和免疫调节功能的影响及其分子机制,探索通过调控细胞自噬和凋亡水平增强机体免疫功能以控制感染进程的新途径,并为后续新药靶位的发现提供理论和实验依据。方法:1.细胞模型的体外实验。以携带spv基因的伤寒沙门菌(Salmonella typhi,ST)ST8为研究对象,用含spv基因的鼠伤寒沙门菌标准毒株SR-11作为阳性对照、不携带spv基因的伤寒沙门菌ST10作为阴性对照,将细菌与小鼠骨髓来源的DCs(bone marrow-derived DCs,BMDCs)共培养;同时用不加细菌的正常细胞作对照组;另设自噬激动剂雷帕霉素(rapamycin,RAPA)干预组,即将细胞预先用含RAPA的培养基过夜处理后分别与上述三种细菌共培养。分别于不同时间段收获细胞,用Western blot法检测BMDCs自噬蛋白Beclin-1的表达,比色法测定其Caspase-3的活性,流式细胞术(flow cytometry,FCM)分析BMDCs的凋亡率、吞噬能力以及表面共刺激分子CD40、CD80和CD86的表达,混合淋巴细胞反应(mixed lymphocyte reaction,MLR)检测BMDCs刺激CD4+和CD8+初始型T(naive T)细胞增殖的能力,ELISA法测定培养液上清中IL-12、IFN-γ、IL-10和IL-4等细胞因子的水平,台盼蓝染色法进行活细胞计数,平板菌落计数法检测BMDCs内活菌数。2.小鼠模型的体内实验。由于伤寒沙门菌是一种严格只对人类致病的病原体,没有合适的动物模型和有效的遗传学工具,本实验利用与其有非常近缘关系的鼠伤寒沙门菌建立动物感染模型,用鼠伤寒沙门菌标准毒株SR-11和不携带spv基因的低毒株BRD509经腹腔注射感染小鼠;同时以未感染细菌的小鼠作为正常对照组;另设RAPA干预组,即在感染模型建立后每天经腹腔注射适量RAPA。分别在不同时间段观察小鼠一般状态,处死小鼠后比较肝脏和脾脏的病理变化,免疫组化法检测肝脏和脾脏Beclin-1和Caspase-3蛋白的表达,FCM分析脾脏DCs的凋亡率、吞噬能力及表面共刺激分子CD40、CD80和CD86的表达,MLR检测脾脏DCs刺激naive T细胞增殖的能力,ELISA法测定血清中IL-12、IFN-γ、IL-10和IL-4等细胞因子的水平,平板菌落计数法检测小鼠肝脏和脾脏的活菌数。结果:1.用RAPA干预前,细菌与BMDCs共作用后2 h,ST10组的Beclin-1表达量高于SR-11组和ST8组,RAPA干预后0 h即可出现此差异;体内实验显示细菌感染早期,BRD509组小鼠脏器的Beclin-1表达高于SR-11组。用RAPA干预后体内外各组Beclin-1的表达均升高。2.用RAPA干预前,体内外各组DCs的凋亡率和Caspase-3活性均随感染时间的延长逐渐升高。体外实验SR-11组和ST8组高于ST10组,用RAPA干预后,SR-11组和ST8组下降,而ST10组上升;体内小鼠脾脏DCs的凋亡率和Caspase-3活性表现为SR-11组高于BRD509组,用药后SR-11组降低,而BRD509组升高。3.用RAPA干预前,随感染时间的延长,体内外实验各组DCs的吞噬能力均不断下降。体外实验ST10组的吞噬率低于SR-11组和ST8组;体内实验BRD509组的吞噬率低于SR-11组。用RAPA干预后,体内外各组DCs的吞噬率均明显下降。4.用RAPA干预前,体内外各组DCs表面共刺激分子CD40、CD80和CD86的表达均随感染时间的延长逐渐升高,刺激naive T细胞增殖的能力也不断增强。体外实验显示ST10组明显强于SR-11组和ST8组;体内实验BRD509组明显强于SR-11组。用RAPA干预后,体内外各组DCs共刺激分子的表达均上升,naive T细胞的增殖程度亦升高。5.用RAPA干预前,体内外各组IL-12和IFN-γ水平随着时间延长显著升高。体外表现为ST10组明显高于SR-11组和ST8组;体内实验BRD509组明显高于SR-11组。用RAPA干预后,体内外各组IL-12和IFN-γ的水平均上升。6.用RAPA干预前,体内外各组在细菌感染早期均未检测到IL-10。体外感染后期SR-11组和ST8组BMDCs分泌的IL-10大量增加,明显高于ST10组;体内感染后期SR-11组的IL-10有表达,而BRD509则仍检测不到。用RAPA干预后,体内外各组IL-10的表达水平均低于用药前。不管RAPA干预与否,各组均未检测到IL-4的表达。7.用RAPA干预前,体外实验SR-11组和ST8组BMDCs的存活率低于ST10组,而胞内活菌数则SR-11组和ST8组高于ST10;体内感染后期,SR-11组小鼠的一般状态较BRD509组差,脏器病理变化严重,活菌数亦较高。用RAPA干预后,体外实验SR-11组和ST8组BMDCs的存活率升高,而ST10组降低,胞内活菌数表现为SR-11组和ST8组降低,而ST10组升高;体内实验SR-11组脏器内活菌数降低而BRD509组升高,SR-11组脏器较用药前损伤减轻而BRD509组加重。结论:1.携带spv基因的沙门菌由于毒力较强,可通过抑制自噬,促进凋亡而加重感染,用RAPA干预其体内外感染模型后自噬被适度激活可对细胞和机体起到一定的保护作用;而无spv基因的沙门菌可通过适度激活自噬以减少细菌对细胞和机体的损害,对自身起到保护作用,当自噬被RAPA过度增强后,反而导致宿主的损伤。2.携带spv基因的沙门菌能抑制小鼠DCs的成熟,使之刺激naive T细胞增殖的能力下降;RAPA可促进DCs的成熟,增强其抗原提呈能力。3.沙门菌感染后刺激小鼠DCs诱导产生以Th1优势应答为主的免疫反应,机体产生的适度免疫应答能有效地清除低毒株的感染,而携带spv基因的沙门菌毒力株能抵抗和逃逸宿主的免疫防御。RAPA可通过增强自噬促进Th1类细胞因子的分泌并抑制Th2类免疫应答从而发挥抗胞内菌感染的作用。更多还原

【Abstract】 Objective: To study the influence of Salmonella plasmid virulence genes (spv) on autophagy, apoptosis and immune function of dendritic cells (DCs), and explore the molecular mechanisms by cell and animal model, respectively. Then search for a new strategy for the control of infection process by regulating autophagy and apoptosis levels to enhance the immune function, and provide theoretical and experimental basis for discovering new drug target sites.Methods:1. The in vitro experiment using a cell model. Salmonella typhi ST8 carrying spv gene is the object of this study. Salmonella typhimurium strain SR-11 containing spv gene was used as a positive control, and Salmonella typhi ST10 as a negative one. Then the 3 strains of Salmonella were co-cultured with mouse bone marrow derived DCs (BMDCs). Another group of BMDCs were treated with an autophagy agonist rapamycin (RAPA) overnight before co-cultured with bacteria, and the uninfected BMDCs were used as normal control group. BMDCs in each group were harvested at different time points, and the autophagy protein Beclin-1 was detected by Western blot; Caspase-3 activity was assayed by colorimetry; the apoptosis rate, endocytosis rate and the expression of co-stimulatory molecules CD40, CD80 and CD86 were determined by flow cytometry (FCM); the proliferation of CD4+ and CD8+ naive T cells stimulated with BMDCs were detected by mixed lymphocyte reaction (MLR); IL-12, IFN-γ, IL-10 and IL-4 secretions were assayed by ELISA; the survival rate of BMDCs was determined by trypan blue stain and the viable bacterial quantities inside BMDCs were detected by plate count method.2. The in vivo experiment using a mouse model. Salmonella typhi only strictly infects human beings and has no animal model. Salmonella typhimurium, a close relative to Salmonella tyhpi, was employed to setup the mouse infection model. Standard Salmonella typhimurium virulence strain SR-11 carrying spv gene were used in the intraperitoneal injection of mouse, and the attenuated strain BRD509 without spv gene was a negative control. Another group of mice were injected with RAPA after bacterial infection and the uninfected mice were used as normal control group. At different time points, the common condition of mouse was obsereved, then the pathological change of livers and spleens were compared after sacrifice of mice; Beclin-1 and Caspase-3 expression in livers and spleens were detected by immunohistochemisty; the apoptosis rate, endocytosis rate and CD40, CD80 and CD86 expression of splenic DCs were determined by FCM; the proliferation of naive T cells stimulated with splenic DCs were detected by MLR; IL-12, IFN-γ, IL-10 and IL-4 secretions in serum were assayed by ELISA, and the viable bacterial amounts in livers and spleens were detected by plate count method.Results:1. Before RAPA intervention, Beclin-1 expression of ST10 group was higher than SR-11 and ST8 group at 2 h post co-culture, and this difference could be observed since 0 h for those Salmonella-infected BMDCs pretreated with RAPA. The in vivo results showed that at the early stage of infection, Beclin-1 expression in organs of BRD509 group was higher than that in SR-11 group. RAPA intervention increased Beclin-1 expression in all groups.2. Before RAPA intervention, the apoptosis rate and Caspase-3 activity of each group increased gradually. The in vitro results showed that SR-11 and ST8 group were higher than ST10 group, and the in vivo results displayed that SR-11 group was higher than BRD509 group. After RAPA treatment, SR-11 and ST8 group were decreased, while ST10 and BRD509 group were increased.3. Before RAPA intervention, the endocytosis activity of DCs in each group was declining with the infection time. ST10 group was lower than SR-11 and ST8 group; BRD509 group was lower than SR-11 group. After intervention with RAPA, BMDCs endocytosis rate of each group were all decreased significantly.4. Before RAPA intervention, CD40, CD80 and CD86 expression on DCs surface and the proliferation of naive T cells stimulated with DCs in all groups were gradually increased; the capability of ST10 group was significantly stronger than SR-11 and ST8 groups; the in vivo BRD509 group was significantly higher than that of SR-11 group. After RAPA intervention, both indexes in each group were increased.5. Before RAPA intervention, all groups’IL-12 and IFN-γlevels were significantly increased, and ST10 group was significantly higher than both SR-11 and ST8 group.The in vivo results showed that BRD509 group was significantly higher than SR -11 group. After RAPA intervention, IL-12 and IFN-γof each group was increased.6. Before RAPA intervention, IL-10 in each group could not detected during the early stage of infection. In the advanced infection stage, IL-10 secretion of BMDCs in SR-11 and ST8 group was detected significantly higher than ST10 group, and IL-10 of SR-11 group in vivo was detected, while BRD509 group still could not be detected. IL-10 expression of each group was decreased after intervented with RAPA. The expression of IL-4 was not detected in any group.7. Before RAPA intervention, the BMDCs’survival rate of SR-11 and ST8 group were lower than ST10 group, but the numbers of intracellular viable bacteria in BMDCs of SR-11 and ST8 group were more than ST10 group. At the advanced stage of infection, mice of SR-11 group showed a worse common condition and more severe pathological changes in organs than BRD509 group, and the viable numbers of bacteria in organs was also higher. After RAPA intervention, the survival rate of BMDCs in SR-11 and ST8 group were increased, while ST10 group reduced, and the viable bacterial numbers in organs of SR-11 and ST8 group were decreased but ST10 group increased. The viable bacteria in organs of SR-11 group were decreased while BRD509 group increased, and the organ injury of SR-11 group was reduced but BRD509 group became more serious.Conclusions:1. Salmonella strains carrying spv gene can promote apoptosis to aggravate the infection by inhibit autophagy. RAPA intervention can protect cells and body by moderate activation of autophagy. Salmonella strains without spv gene can activate autophagy to reduce the bacterial damage to cells and body to protect bacteria themselves. RAPA intervention can lead to host injury when autophagy was hyper-enhanced.2. Salmonella strains carrying spv gene can inhibit DCs maturation to decrease the proliferation of naive T cells. RAPA can promote DCs maturation and enhance the capability of antigen presentation.3. Salmonella infection can stimulate murine DCs to induce a Th1 type immune response. Appropriate immune response can eradicate the infection of attenuated strains. However, spv containing Salmonella strains can resist and escape from immune defense. RAPA can promote the secretion of Th1 type cytokines and suppress Th2 type immune response by enhancing autophagy to strengthen the resistance to Salmonella infection.更多还原