结核分枝杆菌H37Ra和卡介苗感染小鼠及巨噬细胞的研究
发布时间：2019-01-30 15:38
【摘要】：目的:1.了解结核分枝杆菌 H37Ra 和卡介苗(BCG)免疫小鼠后细菌在小鼠体内的定植,及诱导小鼠产生的特异性细胞免疫应答。2.探讨 H37Ra 和 BCG 诱导巨噬细胞产生一氧化氮(NO)的能力,以及细菌在巨噬细胞内生存的能力和破坏巨噬细胞的能力。 方法:1.将 H37Ra 和 BCG 分别皮内接种 BALB/c 小鼠,免疫 15d、30d 及 60d 后,取稀释小鼠脾脏和肺脏匀浆接种罗氏培养基,培养 18d后计 CFU(菌落形成单位);免疫 30d 和 60d 后,取小鼠脾淋巴细胞体外培养并用 PPD 刺激,MTT 法检测脾淋巴细胞转化试验,ELISA 法检测培养上清液中 IFN-γ的产量。2. H37Ra 和 BCG 分别感染 BALB/c 小鼠腹腔巨噬细胞、RAW264.7 细胞株及 THP-1 细胞株, 24h 后取培养上清液,Griess 法检测 NO 的释放量;分别于感染后的 0、4d 及 7d 用 1%TritonX-100 裂解巨噬细胞后接种罗氏培养基,培养 18d 后计 CFU;同时于每个时间点,用 MTT 法检测巨噬细胞存活率的变化。 结果:1.免疫接种后,H37Ra 和 BCG 均可在 BALB/c 小鼠脾脏和肺脏内至少存活 60d。免疫 30d 或 60d 后,脾淋巴细胞刺激指数和 IFN-γ产量检测发现 H37Ra 接种组或 BCG 接种组显著高于未免疫组(P0.05);H37Ra 接种组 IFN-γ产量显著高于 BCG 接种组(P0.05)。2. H37Ra 和 BCG 均可诱导 BALB/c 小鼠腹腔巨噬细胞、RAW264.7 细胞株及 THP-1 细胞株产生多量的 NO,H37Ra 感染组或 BCG 感染组显著高于对照组(P0.05); H37Ra 感染组稍高于 BCG 感染组,但两者无显著性差异(P0.05);H37Ra 和 BCG 均可在巨噬细胞内生存繁殖;H37Ra或 BCG 的感染降低巨噬细胞的存活率,随着感染时间的延长细菌破坏
[Abstract]:Objective: 1. Objective: to investigate the colonization of mycobacterium tuberculosis (H37Ra) and Bacillus Calmette-Guerin (BCG) (BCG) in mice and the specific cellular immune response in mice. 2. To investigate the ability of H37Ra and BCG to induce macrophages to produce nitric oxide (NO), the ability of bacteria to survive in macrophages and the ability to destroy macrophages. Methods: 1. H37Ra and BCG were intradermally inoculated with BALB/c mice respectively. After 15 days and 60 days of immunization, the spleen and lung homogenate of diluted mice were inoculated with Roche medium. After 18 days of culture, CFU (colony forming unit) was counted. After 30 days and 60 days of immunization, mouse splenic lymphocytes were cultured in vitro and stimulated by PPD, spleen lymphocyte transformation test was detected by MTT method, and the production of IFN- 纬 in culture supernatant was detected by ELISA method. The peritoneal macrophages, RAW264.7 cells and THP-1 cell lines of BALB/c mice were infected with H37Ra and BCG, respectively. The supernatants were collected 24 hours later and the release of NO was detected by Griess method. Four and seven days after infection, macrophages were lysed with 1%TritonX-100 and inoculated in Roche medium. After 18 days of culture, the changes of survival rate of macrophages were detected by MTT method at the same time at each time point. Results: 1. After inoculation, both H37Ra and BCG survived for at least 60 days in the spleen and lung of BALB/c mice. After 30 days or 60 days of immunization, the splenic lymphocyte stimulating index and IFN- 纬 production were significantly higher in H37Ra inoculation group or BCG inoculated group than in unimmunized group (P0.05), and IFN- 纬 production in H37Ra inoculated group was significantly higher than that in BCG inoculated group (P0.05). Both H37Ra and BCG could induce peritoneal macrophages in BALB/c mice. The number of NO,H37Ra infection or BCG infection in RAW264.7 cell line and THP-1 cell line was significantly higher than that in control group (P0.05). H37Ra infection group was slightly higher than BCG infection group, but there was no significant difference between them (P0.05); both H37Ra and BCG could survive and reproduce in macrophage; H37Ra or BCG infection decreased the survival rate of macrophage, and with the extension of infection time, bacteria destroyed.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392
本文编号：2418279
[Abstract]:Objective: 1. Objective: to investigate the colonization of mycobacterium tuberculosis (H37Ra) and Bacillus Calmette-Guerin (BCG) (BCG) in mice and the specific cellular immune response in mice. 2. To investigate the ability of H37Ra and BCG to induce macrophages to produce nitric oxide (NO), the ability of bacteria to survive in macrophages and the ability to destroy macrophages. Methods: 1. H37Ra and BCG were intradermally inoculated with BALB/c mice respectively. After 15 days and 60 days of immunization, the spleen and lung homogenate of diluted mice were inoculated with Roche medium. After 18 days of culture, CFU (colony forming unit) was counted. After 30 days and 60 days of immunization, mouse splenic lymphocytes were cultured in vitro and stimulated by PPD, spleen lymphocyte transformation test was detected by MTT method, and the production of IFN- 纬 in culture supernatant was detected by ELISA method. The peritoneal macrophages, RAW264.7 cells and THP-1 cell lines of BALB/c mice were infected with H37Ra and BCG, respectively. The supernatants were collected 24 hours later and the release of NO was detected by Griess method. Four and seven days after infection, macrophages were lysed with 1%TritonX-100 and inoculated in Roche medium. After 18 days of culture, the changes of survival rate of macrophages were detected by MTT method at the same time at each time point. Results: 1. After inoculation, both H37Ra and BCG survived for at least 60 days in the spleen and lung of BALB/c mice. After 30 days or 60 days of immunization, the splenic lymphocyte stimulating index and IFN- 纬 production were significantly higher in H37Ra inoculation group or BCG inoculated group than in unimmunized group (P0.05), and IFN- 纬 production in H37Ra inoculated group was significantly higher than that in BCG inoculated group (P0.05). Both H37Ra and BCG could induce peritoneal macrophages in BALB/c mice. The number of NO,H37Ra infection or BCG infection in RAW264.7 cell line and THP-1 cell line was significantly higher than that in control group (P0.05). H37Ra infection group was slightly higher than BCG infection group, but there was no significant difference between them (P0.05); both H37Ra and BCG could survive and reproduce in macrophage; H37Ra or BCG infection decreased the survival rate of macrophage, and with the extension of infection time, bacteria destroyed.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392
【引证文献】
相关硕士学位论文 前1条
1 王锦彤;结核分枝杆菌Ag85B DNA-H37Ra序贯免疫小鼠的研究[D];重庆医科大学;2006年
，本文编号：2418279
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