【作者】 王健；

【导师】 冷静；

【作者基本信息】 广西医科大学 ， 微生物学， 2005， 硕士

【摘要】 目的: 研究革兰氏阴性非致病菌成团泛菌脂多糖(Pantoea agglomeranslipopolysaccharide,LPSp)作为佐剂促进乙型肝炎表面抗原蛋白(HBsAg)诱导小鼠产生抗-HBs抗体的作用机制。方法: 分离BALB/c小鼠腹腔巨噬细胞,经纯化、鉴定、计数,调整细胞浓度为1×106/ml,在体外分别给予HBsAg加LPSp、HBsAg、LPSp、生理盐水预刺激,体外培养后消化回收各实验组巨噬细胞,进行以下实验(1)将培养48h后的各实验组巨噬细胞按分组经尾静脉注射免疫同系BALB/c小鼠,5天后给予HBsAg 再次免疫,收集初次免疫后15、20、25天的血清,用ELISA法检测乙型肝炎表面抗体(抗-HBsAb)的滴度。(2)观察体外刺激6h后的各实验组巨噬细胞的吞噬功能变化。(3)收集体外刺激48h后的各实验组的巨噬细胞,离心,回收培养上清,检测TNF-α活性和NO浓度。(4)将体外刺激48h后的各实验组的的巨噬细胞按分组经足垫皮下注射免疫同系BALB/c小鼠,5天后收集小鼠腹股沟回流淋巴结,制成细胞悬液,在体外与HBsAg共同培养,用ELISA法检测淋巴细胞培养上清中的IL-4和IFN-γ的含量。结果:(1)LPSp能有效促进HBsAg预致敏的巨噬细胞诱导小鼠抗-HBs抗体产生,其效价在免疫后15d、20d、25d时均较单纯HBsAg致敏组显著增高,差异有统计学意义(P<0.05),而且其抗体效价在免疫15天时已达峰值,在免疫20～25天时下降到一个相对稳定的水平。单纯HBsAg致敏组的抗体效价出现较迟,在免疫后20d时才达高峰。(2)LPSp能刺激巨噬细胞的吞噬功能,单纯LPSp组巨噬细胞吞噬率较NS对照组和单纯HBsAg组的显著增高,差异有统计学意义(P<0.05),与HBsAg加LPSp组比较,差异无统计学意义(P>0.05)。(3)LPSp能刺激体外培养的小鼠腹腔巨噬细胞释放TNF-α和NO,有LPSp刺激的条件下,更多还原

【Abstract】 Objective: To investigate the mechanism of the effect of avirulentPantoea agglomerans lipopolysaccharide (LPSp), that acting as adjuvantfor HBsAg on the production of anti-HBs in mice.Methods: Peritoneal macrophages were harvested from BALB/c miceinjected i.p. with 8ml lps-free RPMI 1640. The peritoneal cells werepurified, identified and counted, then were adjusted to 1×106/ml.Theperitoneal cells were pulsed in vitro with HBsAg+LPSp, HBsAg, LPSp,NS respectively. After cultured in vitro, the adherent macrophages weredigested and collected. (1) BALB/c mice received an i.v. injection ofHBsAg-pulsed peritoneal macrophage,treated or not with LPSp in vitro .Five days later, all mice received a boost of soluble HBsAg i.v. Serawere harvested 15 days, 20 days and 25 days after the first immunization.ELISA determined serum levels of anti-HBs IgG. (2) After peritonealmacrophages were pulsed with HBsAg for 6h, treated or not with LPSp,the phagocytosis function of macrophages were detected. (3) Afterperitoneal macrophage were pulsed with HBsAg, treated or not with LPSp,the culture supernatants were harvested and assaied for TNF-αand NO .(4) HBsAg-pulsed peritoneal macrophage in the presence or absence ofLPSp were injected in the hind footpads of syngeneic mice. Five dayslater, lymph nodes cells were harvested and cultured with HBsAg, IL-4and IFN-γin the supernatants were quantitated by two-site ELISA.Results: (1) The anti-HBs IgG titers from mice immunized withHBsAg-pulsed peritoneal macrophage treated with LPSp were higher thanthose immunized in the absence of LPSp (p<0.05). (2) The phagocytosisrate of LPSp-treated peritoneal macrophages was higher than that ofuntreated macrophages (p<0.05). (3) The levels of TNF-αand NO ofLPSp-treated peritoneal macrophage were higher than that of untreatedmacrophages(p<0.05) . (4) HBsAg-pulsed peritoneal cells in thepresence of LPSp could induce higher production of IL-4 than which inthe absence of LPSp (p<0.05).Conclusion: LPSp could act as the adjuvant of HBsAg, andeffectively increase the anti-HBS in mice immunized with HBsAg .Themechanism could be: (1) LPSp could activate macrophages.①increasingthe phagocytosis function of macrophages. ②stimulating the productionof TNF-αand NO of macrophages.(2) LPSp could stimulate HBsAg-pulsedmacrophages to induce production of IL-4 of lymph nodes cells,suggesting that LPSp could stimulate HBsAg-pulsed macrophages toinduce the activation of Th2-type lymphocyte and helping the productionof anti-HBS. (3) LPSp couldn’t stimulate the production of IFN-γinduced by HBsAg-pulsed macrophages, suggesting that LPSp couldn’tstimulate the activation of Th1-type lymphocyte induced byHBsAg-pulsed macrophages.更多还原