【摘要】：目的:1.了解結(jié)核分枝桿菌 H37Ra 和卡介苗(BCG)免疫小鼠后細(xì)菌在小鼠體內(nèi)的定植,及誘導(dǎo)小鼠產(chǎn)生的特異性細(xì)胞免疫應(yīng)答。2.探討 H37Ra 和 BCG 誘導(dǎo)巨噬細(xì)胞產(chǎn)生一氧化氮(NO)的能力,以及細(xì)菌在巨噬細(xì)胞內(nèi)生存的能力和破壞巨噬細(xì)胞的能力。 方法:1.將 H37Ra 和 BCG 分別皮內(nèi)接種 BALB/c 小鼠,免疫 15d、30d 及 60d 后,取稀釋小鼠脾臟和肺臟勻漿接種羅氏培養(yǎng)基,培養(yǎng) 18d后計(jì) CFU(菌落形成單位);免疫 30d 和 60d 后,取小鼠脾淋巴細(xì)胞體外培養(yǎng)并用 PPD 刺激,MTT 法檢測(cè)脾淋巴細(xì)胞轉(zhuǎn)化試驗(yàn),ELISA 法檢測(cè)培養(yǎng)上清液中 IFN-γ的產(chǎn)量。2. H37Ra 和 BCG 分別感染 BALB/c 小鼠腹腔巨噬細(xì)胞、RAW264.7 細(xì)胞株及 THP-1 細(xì)胞株, 24h 后取培養(yǎng)上清液,Griess 法檢測(cè) NO 的釋放量;分別于感染后的 0、4d 及 7d 用 1%TritonX-100 裂解巨噬細(xì)胞后接種羅氏培養(yǎng)基,培養(yǎng) 18d 后計(jì) CFU;同時(shí)于每個(gè)時(shí)間點(diǎn),用 MTT 法檢測(cè)巨噬細(xì)胞存活率的變化。 結(jié)果:1.免疫接種后,H37Ra 和 BCG 均可在 BALB/c 小鼠脾臟和肺臟內(nèi)至少存活 60d。免疫 30d 或 60d 后,脾淋巴細(xì)胞刺激指數(shù)和 IFN-γ產(chǎn)量檢測(cè)發(fā)現(xiàn) H37Ra 接種組或 BCG 接種組顯著高于未免疫組(P0.05);H37Ra 接種組 IFN-γ產(chǎn)量顯著高于 BCG 接種組(P0.05)。2. H37Ra 和 BCG 均可誘導(dǎo) BALB/c 小鼠腹腔巨噬細(xì)胞、RAW264.7 細(xì)胞株及 THP-1 細(xì)胞株產(chǎn)生多量的 NO,H37Ra 感染組或 BCG 感染組顯著高于對(duì)照組(P0.05); H37Ra 感染組稍高于 BCG 感染組,但兩者無顯著性差異(P0.05);H37Ra 和 BCG 均可在巨噬細(xì)胞內(nèi)生存繁殖;H37Ra或 BCG 的感染降低巨噬細(xì)胞的存活率,隨著感染時(shí)間的延長(zhǎng)細(xì)菌破壞
[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- 緯 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- 緯 production were significantly higher in H37Ra inoculation group or BCG inoculated group than in unimmunized group (P0.05), and IFN- 緯 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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R392

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 王錦彤;結(jié)核分枝桿菌Ag85B DNA-H37Ra序貫免疫小鼠的研究[D];重慶醫(yī)科大學(xué);2006年

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本文編號(hào)：2418279