表达幽门螺杆菌粘附素保守区的减毒鼠伤寒沙门菌免疫治疗作用的研究

Study on Immunization Treatment of The Attenuated Salmonella typhimurium Strain Expressing Conservative Region of Helicobacter pylori Adhesin

探讨表达幽门螺杆菌 (Helicobacterpylori,Hp)粘附素保守区 (AB)的减毒鼠伤寒沙门菌X4 0 72 (pYA2 4 8 AB)的免疫治疗效果 ,以及可能的免疫治疗机制 ,以确定X4 0 72 (pYA2 4 8 AB)在Hp疫苗研制中的应用价值 .建立Hp感染小鼠模型 ,在该模型中评价X4 0 72 (pYA2 4 8 AB)治疗Hp感染的效果 ,运用细菌培养法观察Hp根除率及定植量的改变 ,流式细胞术分析免疫后小鼠脾细胞亚型 ,IL 2和IL 4细胞依赖株的细胞测活法检测细胞因子 ,ELISA法检测小鼠血清及肠液中抗AB抗体产生情况 .结果表明 ,免疫治疗后疫苗治疗组根除率为 5 3 3% ,显著高于X4 0 72 (pYA2 4 8)和PBS对照组 (P <0 0 1 ) .未根除Hp的小鼠 ,疫苗治疗组Hp的定植密度明显低于其他两组 (P <0 0 1 ) .应用流式细胞仪分析免疫小鼠脾CD4 + /CD8+ T淋巴细胞的比值时 ,发现疫苗治疗组和鼠伤寒菌对照组的比值均显著高于PBS对照组 (P <0 0 5 ) ,而疫苗治疗组的比值又显著高于鼠伤寒菌对照组 (P <0 0 5 ) .进一步对细胞因子进行分析发现 ,与PBS对照组相比免疫治疗组和鼠伤寒菌对照组IL 2和IL 4明显升高(P <0 0 5 ) .同时 ,免疫治疗组与鼠伤寒菌对照组相比 ,IL 4增高明显 (P <0 0 5 ) .针对AB的抗体测定结果发现 。

In order to investigate the treatment efficacy and the potenti al mechanism of the attenuated Salmonella typhimurium strain expressing conser vative region(AB) of Helicobacter pylori ( Hp ) adhesion X4072(pYA248 AB) in mouse model infected with Hp , the feasibility of X4072(pYA248 AB) in Hp vaccination was determined. Hp infected mouse model was established and applied in oral vaccination. The density of bacterial colonization was determined by the semi quantitative bacterial culture assay. The T cell subsets of spleenocytes were assayed by flow cytometry (FCM) after immunization. IL 2 and IL 4 were detected through MTT. Mice sera and intestinal fluid were separatively tested for AB specific IgG and IgA by ELISA. The results showed that after immune treatment, the eradicate rate of vaccine treatment group was 53 3%, which was significantly higher than that of other two control groups (X4072(pYA248 AB) vs X4072(pYA248) and PBS, P <0 01). The Hp colonized density of vaccination group with Hp infection was significant ly lower than that of other two groups (X4072(pYA248 AB) vs X4072(pYA248) and PBS, P <0 01). Comparing the ratio of spleen CD4+/CD8+ T lymphocytic cells of the immuned mice by flow cytometer, it was found that the ratio of vaccine treatment group and mice typhoid fever germ control group were significantly higher than that of negative control group (X4072(pYA248 AB) and X4072(pYA248) vs PBS, P <0 05) respectively. In addition, the ratio of vaccine treatment was significantly higher than that of mice typhoid fever germ control group (X4072(pYA248 AB) vs X4072(pYA248), P <0 05). The further analysis of cytokine it was found that IL 2 and IL 4 of vaccine treatment group and mice typhoid fever germ control group were significantly higher than those of negati ve control group (X4072(pYA248 AB) and X4072(pYA248) and PBS, P <0 05) resp ectively, meanwhile, the IL 4 of vaccine treatment group was significantly highe r than that of mice typhoid fever germ control group (X4072(pYA248) vs PBS, P <0 05). Humoral immunity analysis showed that IgA was only detected in the immune treatment group, and the IgG antibody titer of immune treatment group increased significantly than that of the other two group, and the titer reached 1∶10 000 two weeks after the second i ntensive immunization. By the animal experiments it is suggested that X4072(pYA248 AB)can eradicate or significantly reduce the Hp colonization on the mice stomach. The potential immune therapeutic mechanism included the increase of CD4+/CD8+ ratio, induction of Th1 and Th2 response, and production of the specific antibodies.