【摘要】：目的 研究從解脲脲原體(Uu)提取的脂質(zhì)相關(guān)膜蛋白(LAMPs)在體外誘導(dǎo)小鼠巨噬細胞表達誘導(dǎo)性一氧化氮合酶(iNOS)及產(chǎn)生一氧化氮(NO)的水平,研究該膜蛋白能否激活小鼠巨噬細胞中核因子kappa B(NF-κB)及誘導(dǎo)細胞發(fā)生凋亡的情況,以便了解Uu潛在的致病性,為進一步探討Uu LAMPs誘導(dǎo)小鼠巨噬細胞表達iNOS及誘導(dǎo)巨噬細胞發(fā)生凋亡的分子機制提供實驗依據(jù)。 方法 用從Uu提取的LAMPs刺激小鼠巨噬細胞,用Griess試劑測定經(jīng)刺激后的小鼠巨噬細胞產(chǎn)生的NO水平,以RT-PCR和Western blot方法分析iNOS的表達水平,用細胞免疫組化、間接免疫熒光檢測NF-κB的激活,用Western blot檢測核提取物中NF-κB蛋白的表達,且利用RT-PCR和Western blot方法檢測NF-κB的特異性抑制劑二硫代氨基甲酸吡咯烷(PDTC)和蛋白酶抑制劑放線菌酮(CHX)對iNOS的表達及對NF-κB激活的影響。用細胞凋亡檢測試劑盒檢測經(jīng)Uu LAMPs處理的小鼠巨噬細胞的凋亡情況。 結(jié)果 Uu LAMPs能誘導(dǎo)小鼠巨噬細胞表達iNOS,且能以時間和劑量依賴方式刺激小鼠巨噬細胞產(chǎn)生NO,當(dāng)LAMPs的濃度為3μg/mL時產(chǎn)生的NO量最多,為13.56±0.5μmol/L;但當(dāng)LAMPs從3μg/mL增加到5μg/mL時,NO產(chǎn)生的量反而減少。另外當(dāng)LAMPs刺激細胞4h后即可從培養(yǎng)基中檢測到NO,而刺激32h后產(chǎn)生的NO量則達到高峰。在用該膜蛋白與NF-κB的抑制劑PDTC或蛋白酶抑制劑
[Abstract]:Objective To study the expression of nitric oxide synthase (iNOS) and the level of nitric oxide (NO) in mouse macrophages induced by Uu in vitro. whether the membrane protein can activate the nuclear factor kappab (NF-B) in the mouse macrophages and induce the apoptosis of the cells in order to understand the potential pathogenicity of the Uu, In order to further study the mechanism of Uu LMPs to induce the expression of iNOS in mouse macrophages and induce the apoptosis of macrophages, the experimental basis was provided. Methods The mouse macrophages were stimulated with LAMs extracted from Uu, and the level of NO produced by the stimulated mouse macrophages was determined by Griess reagent. The expression level of iNOS was analyzed by RT-PCR and Western blot, and the expression level of iNOS was detected by immunohistochemistry and indirect immunofluorescence. The expression of NF-EMAB in the nuclear extract was detected by Western blot and the expression of the specific inhibitor of NF-EMAB by RT-PCR and Western blot was detected by Western blot.-The effect of the activation of the antigen-B. The mice treated with the Uu LAMPs were tested with a cell apoptosis assay kit. Results Uu LMPs can induce the expression of iNOS in mouse macrophages, and can stimulate the macrophage to produce NO in time and dose-dependent manner. When the concentration of LAMs is 3. m from 3 & mu; g/ mL to 5 in mu. g/ ml, the amount of NO was reduced, and no NO was detected from the medium after the LAMs stimulated the cell for 4 h. The amount of NO produced after stimulation of 32h reached a peak. The membrane protein and NF were used.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R375

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