本文選題：結(jié)核分枝桿菌 + 樹突狀細(xì)胞　； 參考：《大理大學(xué)》2017年碩士論文

【摘要】：目的建立Mtb感染小鼠DC2.4細(xì)胞模型,探討不同毒力Mtb誘導(dǎo)小鼠DC2.4細(xì)胞凋亡的差異及相關(guān)分子機(jī)制,并研究TRL4-NOD2 (T4N2)協(xié)同信號傳遞靶向增強(qiáng)DC的活性以及活化的DC對Mtb生長的抑制率,進(jìn)而分析DC在抗結(jié)核感染中的機(jī)制,為研究結(jié)核的致病機(jī)制及新型抗結(jié)核的免疫療法提供新的線索和理論依據(jù)。方法分別采用結(jié)核分枝桿菌標(biāo)準(zhǔn)減毒株(H37Ra株)、大理臨床分離株(14-11株)感染小鼠DC2.4細(xì)胞,建立小鼠DC2.4細(xì)胞體外Mtb感染模型,采用Annexin V-FITC /PI熒光標(biāo)記流式細(xì)胞術(shù)依次檢測感染后五個不同時間段小鼠DC 2.4的細(xì)胞凋亡情況,并分析其差異表達(dá)。分別用TLR4配體(LPS)、NOD2配體(MDP)和TLR4-NOD2 (T4N2)雙配體刺激小鼠DC2.4后,酶聯(lián)免疫吸附試驗(ELISA)法定量檢測不同時間段上清液中IL-6和IL-12的分泌情況。用Mtb感染DC 4h后,再分別用單配體LPS、MDP以及T4N2雙配體刺激培養(yǎng)24h,收集細(xì)胞并裂解后接種于羅氏培養(yǎng)基,3周后觀察并計數(shù)菌落生長數(shù),計算細(xì)菌生長的抑制率。實驗數(shù)據(jù)應(yīng)用SPSS17.0統(tǒng)計軟件進(jìn)行統(tǒng)計學(xué)分析。結(jié)果1.結(jié)核分枝桿菌標(biāo)準(zhǔn)減毒株H37Ra與大理臨床分離株14-11均能誘導(dǎo)小鼠DC2.4細(xì)胞凋亡,在感染后6h凋亡率升高明顯,兩組細(xì)胞比較凋亡率差異有統(tǒng)計學(xué)意義(P0.05)。大理臨床分離株14-11誘導(dǎo)小鼠DC2.4細(xì)胞凋亡率在誘導(dǎo)后各時間點均低于標(biāo)準(zhǔn)減毒株H37Ra誘導(dǎo)的凋亡率,誘導(dǎo)后6h凋亡率升高顯著。2. LPS配體刺激組、MDP配體刺激組及T4N2雙配體刺激組分別刺激小鼠DC2.4后均能誘導(dǎo)IL-6、IL-12細(xì)胞因子釋放,T4N2雙信號刺激組與空白組、LPS組、MDP組比較細(xì)胞因子IL-6 (P0.01)和IL-12 (P0.01)分泌增多,在不同時間段均存在差異(P 0.01)。3.TLR4 配體(LPS)、NOD2 配體(MDP)和 T4N2 雙配體(LPS+MDP)組分別刺激小鼠DC2.4后,Mtb的生長均有不同程度的抑制,T4N2雙配體活化的DC2.4對Mtb的生長抑制率較對照組及單配體刺激組明顯增強(qiáng)。結(jié)論1.小鼠DC2.4細(xì)胞被不同毒力的Mtb感染后均能快速誘導(dǎo)其凋亡,凋亡率與所誘導(dǎo)結(jié)核菌株的不同毒力有關(guān),毒力較弱的H37Ra標(biāo)準(zhǔn)減毒株較毒力較強(qiáng)的大理臨床分離株14-11誘導(dǎo)的凋亡率更高。2. TLR4-NOD2協(xié)同信號傳遞能增強(qiáng)DC的活化。3.活化后的DC可抑制Mtb的生長。
[Abstract]:Objective to establish a mouse DC2.4 cell model infected with Mtb, to explore the difference of apoptosis induced by different virulence Mtb and its molecular mechanism, and to study the synergistic signal transduction targeting enhancement of DC activity and the inhibitory rate of activated DC on Mtb growth. The mechanism of DC in anti-tuberculosis infection is analyzed, which provides a new clue and theoretical basis for studying the pathogenetic mechanism of tuberculosis and new anti-tuberculosis immunotherapy. Methods mice DC2.4 cells were infected with Mycobacterium tuberculosis standard attenuated strain H37Ra and Dali clinical isolates 14-11, respectively. The model of Mtb infection of mouse DC2.4 cells in vitro was established. Annexin V-FITC / Pi fluorescence labeling flow cytometry was used to detect the apoptosis of DC 2. 4 cells in five different time periods after infection, and the differential expression of DC 2. 4 cells was analyzed. After the mice were stimulated with TLR4 ligands (TLR4) and TLR4-NOD2 T4N _ 2), the secretion of IL-6 and IL-12 in supernatant at different time periods was assayed by Elisa. After the DC was infected with Mtb for 4 h, the single ligand LPSN MDP and the double ligand T4N2 were used to stimulate culture for 24 h respectively. The cells were collected and lysed and inoculated in Roche medium for 3 weeks. The colony growth number was observed and counted, and the inhibition rate of bacterial growth was calculated. The experimental data were analyzed by SPSS17.0 statistical software. Result 1. Mycobacterium tuberculosis standard attenuated strain H37Ra and Dali clinical isolate 14-11 could induce apoptosis of DC2.4 cells in mice. The apoptotic rate increased significantly at 6 h after infection. There was significant difference in apoptosis rate between the two groups (P 0.05). The apoptosis rate of DC2.4 cells induced by Dali clinical isolates 14-11 was lower than that induced by standard attenuated strain H37Ra at all time points after induction, and the apoptotic rate increased significantly at 6 h after induction. The LPS ligand stimulation group and the T4N2 double ligand stimulation group could induce the release of IL-6 and IL-12 cytokines after stimulation of DC2.4 in mice respectively. The cytokine levels of IL-6 P0.01) and IL-12 P0.01) were increased in the T4N2 double signal stimulation group compared with those in the control group. At different time points, there were differences in the growth inhibition rate of Mtb induced by P 0.01).3.TLR4 ligands and T4N2 double ligand ligands, respectively. The growth of Mtb was inhibited by DC2.4 activated by T4N2 ligands in different degrees compared with the control group (P < 0.05), and the growth inhibition rate of Mtb was higher than that of the control group (P < 0.05), and the growth inhibition rate of Mtb was significantly higher than that of the control group (P < 0.05). The single ligand stimulation group was significantly enhanced. Conclusion 1. Mouse DC2.4 cells were infected with Mtb with different virulence, and the apoptosis rate was related to the virulence of tuberculous strains. The rate of apoptosis induced by H37Ra standard attenuated strain with lower virulence was higher than that of Dali clinical isolate with stronger virulence. TLR4-NOD2 synergistic signal transduction can enhance DC activation. 3. Activated DC could inhibit the growth of Mtb.
【學(xué)位授予單位】：大理大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R52

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