树突状细胞抗结核分枝杆菌感染机制的初步研究

发布时间：2018-05-30 12:17

本文选题：结核分枝杆菌 + 树突状细胞　；参考：《大理大学》2017年硕士论文

【摘要】：目的建立Mtb感染小鼠DC2.4细胞模型,探讨不同毒力Mtb诱导小鼠DC2.4细胞凋亡的差异及相关分子机制,并研究TRL4-NOD2 (T4N2)协同信号传递靶向增强DC的活性以及活化的DC对Mtb生长的抑制率,进而分析DC在抗结核感染中的机制,为研究结核的致病机制及新型抗结核的免疫疗法提供新的线索和理论依据。方法分别采用结核分枝杆菌标准减毒株(H37Ra株)、大理临床分离株(14-11株)感染小鼠DC2.4细胞,建立小鼠DC2.4细胞体外Mtb感染模型,采用Annexin V-FITC /PI荧光标记流式细胞术依次检测感染后五个不同时间段小鼠DC 2.4的细胞凋亡情况,并分析其差异表达。分别用TLR4配体(LPS)、NOD2配体(MDP)和TLR4-NOD2 (T4N2)双配体刺激小鼠DC2.4后,酶联免疫吸附试验(ELISA)法定量检测不同时间段上清液中IL-6和IL-12的分泌情况。用Mtb感染DC 4h后,再分别用单配体LPS、MDP以及T4N2双配体刺激培养24h,收集细胞并裂解后接种于罗氏培养基,3周后观察并计数菌落生长数,计算细菌生长的抑制率。实验数据应用SPSS17.0统计软件进行统计学分析。结果1.结核分枝杆菌标准减毒株H37Ra与大理临床分离株14-11均能诱导小鼠DC2.4细胞凋亡,在感染后6h凋亡率升高明显,两组细胞比较凋亡率差异有统计学意义(P0.05)。大理临床分离株14-11诱导小鼠DC2.4细胞凋亡率在诱导后各时间点均低于标准减毒株H37Ra诱导的凋亡率,诱导后6h凋亡率升高显著。2. LPS配体刺激组、MDP配体刺激组及T4N2双配体刺激组分别刺激小鼠DC2.4后均能诱导IL-6、IL-12细胞因子释放,T4N2双信号刺激组与空白组、LPS组、MDP组比较细胞因子IL-6 (P0.01)和IL-12 (P0.01)分泌增多,在不同时间段均存在差异(P 0.01)。3.TLR4 配体(LPS)、NOD2 配体(MDP)和 T4N2 双配体(LPS+MDP)组分别刺激小鼠DC2.4后,Mtb的生长均有不同程度的抑制,T4N2双配体活化的DC2.4对Mtb的生长抑制率较对照组及单配体刺激组明显增强。结论1.小鼠DC2.4细胞被不同毒力的Mtb感染后均能快速诱导其凋亡,凋亡率与所诱导结核菌株的不同毒力有关,毒力较弱的H37Ra标准减毒株较毒力较强的大理临床分离株14-11诱导的凋亡率更高。2. TLR4-NOD2协同信号传递能增强DC的活化。3.活化后的DC可抑制Mtb的生长。
[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.
【学位授予单位】：大理大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R52

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