下调DC-SIGN分子对下游抗结核免疫应答影响的实验研究

发布时间：2018-01-06 11:37

  本文关键词：下调DC-SIGN分子对下游抗结核免疫应答影响的实验研究　出处：《重庆医科大学》2010年硕士论文　论文类型：学位论文

  更多相关文章： 树突状细胞 ManLAM DC-SIGN RNA干扰 初始CD4~+ CD45RA~+T细胞 结核病

【摘要】： 目的:探讨下调人树突状细胞特异性细胞间粘附分子-3-结合非整合素(dendritic cell - specific ICAM - 3 grabbing nonintergrin, DC-SIGN)分子对下游抗结核免疫应答的影响,进而探讨DC-SIGN在宿主抗结核免疫中的作用。 方法:1. ManLAM对DCs成熟及功能的影响:密度梯度离心分离人外周血单个核细胞,贴壁2h去除悬浮细胞后,用rhGM-CSF和rhIL-4刺激诱导分化为DCs。第六天实验分为DCs组和ManLAM组,加ManLAM和LPS刺激。第七天收集细胞,流式细胞仪检测DC-SIGN、HLA-DR、CD83和CD86表达水平,ELISA检测上清液中IL-12,IL-10水平。 2.下调DC-SIGN后对DCs成熟的影响:①RNAi效果检测:分三组即DCs组、RNAi组和阴性对照组,加慢病毒载体共孵育12h后换液,继续培养60h后用荧光显微镜观察转染效率及荧光强度,第六天加LPS刺激。第七天收集细胞,RT-PCR检测DC-SIGN mRNA水平,Western Blot检测DC-SIGN蛋白表达水平;②DCs转染后表面分子表达及细胞因子水平变化:分四组即DCs组、RNAi组、ManLAM组和ManLAM~+RNAi组,流式细胞仪检测DCs表面分子表达水平,ELISA检测IL-12,IL-10水平。 3.下调DC-SIGN后对下游初始T细胞活化增殖的影响:初始CD4~+CD45RA~+T细胞与DCs共培养,用ELISA检测培养上清液中IFN-γ水平。混合淋巴细胞反应检测各组DCs对CD4~+T淋巴细胞刺激增殖能力的影响。 4.下调DC-SIGN后对巨噬细胞活化吞噬杀灭结核杆菌的影响:用与DCs共培养后的初始CD4~+CD45RA~+T细胞与培养9天后的巨噬细胞混合培养,48h后检测TNF-α水平和NO水平。透射电镜观察巨噬细胞吞噬结核杆菌情况,裂解巨噬细胞并接种培养,一周后计数。 结果:1.外周血单核细胞诱导分化的DCs在培养的第7天出现典型的形态学特征。流式细胞仪检测ManLAM组表达CD83,CD86,DC-SIGN水平低于DCs组(p0.05)。ELISA检测ManLAM组IL-12水平低于DCs组,IL-10水平高于DCs组(p0.05)。 2.①荧光显微镜下显示各个转染组的细胞表面出现绿色荧光,当感染复数(MOI)为20时,DCs感染病毒的效率达85%。RT-PCR证实RNA干扰后DC-SIGN mRNA表达水平显著低于阴性对照组和DCs组,差异有统计学意义(p0.05)。Western Blot检测干扰组DCs内总DC-SIGN水平显著低于阴性对照组和DCs组,差异有统计学意义(p0.05)。②流式细胞仪检测DC-SIGN阳性率在RNAi组、ManLAM组及ManLAM~+RNAi组与DCs组相比显著下调,差异有统计学意义(p0.05);各组间HLA-DR阳性率相比较差异无显著性(p0.05)。ManLAM组CD83和CD86表达低于其余各组(p0.05);其余各组间相比差异无统计学意义(p0.05)。ELISA检测显示ManLAM组IL-10表达水平高于其他组(p0.05),ManLAM+RNAi组高于DCs组及RNAi组(p0.05),其余各组间差异无统计学意义(p0.05);ManLAM组IL-12表达水平低于其他各组(p0.05),其余各组差异无统计学意义(p0.05)。 3. ELISA检测DCs与初始CD4~+ CD45RA~+T细胞共培养液中IFN-γ水平显示,ManLAM组低于其余各组(p0.05);ManLAM+RNAi组IFN-γ水平低于DCs组(p0.05);其余各组间差异无统计学意义(p0.05)。混合淋巴细胞反应检测显示ManLAM组CPM值低于其余各组(p0.05),其余各组间差异无统计学意义(p0.05)。 4. ELISA检测T细胞与巨噬细胞共培养液中TNF-α水平显示,ManLAM组TNF-α水平低于DCs组(p0.05),ManLAM+RNAi组TNF-α水平高于ManLAM组(p0.05);硝酸还原法检测NO水平显示,ManLAM组NO水平低于DCs组(p0.05),ManLAM+RNAi组NO水平高于ManLAM组(p0.05)。巨噬细胞内活菌计数显示,ManLAM组高于DCs组(p0.05),ManLAM+RNAi组活菌量低于ManLAM组(p0.05)。 结论: 1. ManLAM能干扰DCs成熟,下调DCs诱导的淋巴细胞增殖能力和激活初始CD4~+CD45RA~+T细胞向Th1细胞分化能力,降低巨噬细胞活化,最终导致巨噬细胞杀灭结核杆菌能力降低。 2.慢病毒介导的RNA干扰能有效的抑制DCs表面DC-SIGN分子表达,DC-SIGN表达降低对DCs成熟度及DCs诱导的下游免疫功能无影响。 3.慢病毒介导的RNA干扰人DC-SIGN表达能恢复ManLAM对DCs成熟的抑制及DCs诱导的下游免疫功能,最终恢复或部分恢复下游巨噬细胞杀灭MTB的能力。
[Abstract]:Objective: To investigate the downregulation of human dendritic cell specific intercellular adhesion molecule -3- (dendritic cell combined with non integrin - specific ICAM - grabbing 3 nonintergrin, DC-SIGN) effect on molecular downstream anti TB immune response, and then explore the DC-SIGN in the host anti TB immune response.
Methods: 1. effects of ManLAM on the function of DCs and isolation of human peripheral blood mononuclear cells by density gradient centrifugation and adherent 2H removal of suspended cells, induced differentiation of DCs. in the sixth day experiment was divided into DCs group and ManLAM group by rhGM-CSF and rhIL-4 stimulation, plus ManLAM and LPS stimulation. Seventh days were collected for detection DC-SIGN, flow cytometry, HLA-DR, CD83 and the expression of CD86, IL-12, ELISA detected IL-10 level.
2. effect of down-regulation of DC-SIGN in mature DCs: detection of RNAi effect: divided into three groups: DCs group, RNAi group and negative control group, and the lentiviral vectors were co incubated with 12h after the change of liquid, continue to culture 60H was observed by fluorescence microscopy and fluorescence intensity of transfection efficiency, sixth days LPS seventh days to collect cell stimulation. RT-PCR, detection of DC-SIGN mRNA level, the expression level of DC-SIGN protein was detected by Western Blot; expression of surface molecules of DCs after transfection and cytokine levels were divided into four groups: DCs group, RNAi group, ManLAM group and ManLAM~+RNAi group. The detection of DCs surface expression level of flow cytometric detection of IL-12, ELISA, IL-10 level.
3. the effect of down regulation of DC-SIGN on the activation and proliferation of downstream T cells. The initial CD4~+CD45RA~+T cells were co cultured with DCs, and IFN- was detected by ELISA. The effect of DCs on CD4~+T lymphocyte stimulated proliferation was detected by mixed lymphocyte reaction.
4. down after DC-SIGN activation effect on macrophage phagocytic killing of Mycobacterium tuberculosis by cultivation and initial CD4~+CD45RA~+T cells were co cultured with DCs after 9 days of macrophage co culture, detection of TNF- alpha and NO level after 48h. TEM observation of macrophage phagocytosis of Mycobacterium tuberculosis, cultured macrophages and cracking inoculation, a week after the count.
Results: 1. peripheral blood mononuclear cells induced differentiation of DCs typical morphological features in cultured seventh days. Flow cytometry was used to detect the expression of CD83 in group ManLAM, CD86, DC-SIGN level lower than that of group DCs (P0.05).ELISA ManLAM detection of IL-12 group were lower than DCs group, the level of IL-10 is higher than that of DCs group (P0.05).
2. under the fluorescent microscope showed green fluorescence appeared in each transfection group cells, when the multiplicity of infection (MOI) is 20, the efficiency of DCs infected 85%.RT-PCR RNA confirmed after interference DC-SIGN expression level of mRNA was significantly lower than the negative control group and DCs group, the difference was statistically significant (P0.05).Western Blot detection DCs interference group the total DC-SIGN level was significantly lower than the negative control group and DCs group, the difference was statistically significant (P0.05). The positive rate of flow cytometry in DC-SIGN RNAi group, ManLAM group and ManLAM~+RNAi group compared with DCs group were significantly reduced, the difference was statistically significant between the groups (P0.05); the positive rate of HLA-DR was significantly the expression of.ManLAM (P0.05) of group CD83 and CD86 was lower than that of other groups (P0.05); other groups had no significant difference (P0.05).ELISA showed that the expression level of ManLAM in group IL-10 was higher than other groups (P0.05, ManLAM+R) Group NAi was higher than group DCs and group RNAi (P0.05), the difference between the other groups was not statistically significant (P0.05), the expression level of IL-12 in ManLAM group was lower than that in other groups (P0.05), and there was no significant difference between the other groups (P0.05).
3. ELISA initial CD4~+ detection of DCs and CD45RA~+T cells were co cultured in liquid IFN- levels showed that ManLAM group was lower than that of other groups (P0.05); group ManLAM+RNAi IFN- levels were lower than that of DCs group (P0.05); no significant difference among other groups (P0.05). The mixed lymphocyte reaction test showed that ManLAM group CPM was lower than the other groups (P0.05), there was no significant difference among other groups (P0.05).
4. ELISA detection of T cells and macrophages were cultured in liquid TNF- levels showed that ManLAM group TNF- a level lower than that of DCs group (P0.05), ManLAM+RNAi group TNF- a level higher than that of ManLAM group (P0.05); NO was used to detect the level of nitrate reduction method showed that the NO level of ManLAM group was lower than that of DCs group (P0.05), NO level of ManLAM+RNAi group was higher than that of ManLAM group (P0.05). Intracellular viable count showed that ManLAM group than in DCs group (P0.05), ManLAM+RNAi group of viable bacteria was lower than that of ManLAM group (P0.05).
Conclusion:
1. ManLAM can interfere with DCs maturation, down regulate DCs induced lymphocyte proliferation and activate CD4~+CD45RA~+T cells to differentiate into Th1 cells, reduce macrophage activation, and ultimately lead to the decrease of macrophage killing ability of Mycobacterium tuberculosis.
2. lentivirus mediated RNA interference can effectively inhibit the expression of DC-SIGN on DCs surface, and the decrease of DC-SIGN expression has no effect on DCs maturity and DCs induced downstream immune function.
3., lentivirus mediated RNA interference on human DC-SIGN expression can restore the inhibition of ManLAM on DCs maturation and DCs induced downstream immune function, and ultimately restore or partly restore the ability of downstream macrophages to destroy MTB.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392.12

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