本文關(guān)鍵詞：下調(diào)DC-SIGN分子對下游抗結(jié)核免疫應(yīng)答影響的實驗研究　出處：《重慶醫(yī)科大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 樹突狀細(xì)胞 ManLAM DC-SIGN RNA干擾 初始CD4~+ CD45RA~+T細(xì)胞 結(jié)核病

【摘要】： 目的:探討下調(diào)人樹突狀細(xì)胞特異性細(xì)胞間粘附分子-3-結(jié)合非整合素(dendritic cell - specific ICAM - 3 grabbing nonintergrin, DC-SIGN)分子對下游抗結(jié)核免疫應(yīng)答的影響,進(jìn)而探討DC-SIGN在宿主抗結(jié)核免疫中的作用。 方法:1. ManLAM對DCs成熟及功能的影響:密度梯度離心分離人外周血單個核細(xì)胞,貼壁2h去除懸浮細(xì)胞后,用rhGM-CSF和rhIL-4刺激誘導(dǎo)分化為DCs。第六天實驗分為DCs組和ManLAM組,加ManLAM和LPS刺激。第七天收集細(xì)胞,流式細(xì)胞儀檢測DC-SIGN、HLA-DR、CD83和CD86表達(dá)水平,ELISA檢測上清液中IL-12,IL-10水平。 2.下調(diào)DC-SIGN后對DCs成熟的影響:①RNAi效果檢測:分三組即DCs組、RNAi組和陰性對照組,加慢病毒載體共孵育12h后換液,繼續(xù)培養(yǎng)60h后用熒光顯微鏡觀察轉(zhuǎn)染效率及熒光強度,第六天加LPS刺激。第七天收集細(xì)胞,RT-PCR檢測DC-SIGN mRNA水平,Western Blot檢測DC-SIGN蛋白表達(dá)水平;②DCs轉(zhuǎn)染后表面分子表達(dá)及細(xì)胞因子水平變化:分四組即DCs組、RNAi組、ManLAM組和ManLAM~+RNAi組,流式細(xì)胞儀檢測DCs表面分子表達(dá)水平,ELISA檢測IL-12,IL-10水平。 3.下調(diào)DC-SIGN后對下游初始T細(xì)胞活化增殖的影響:初始CD4~+CD45RA~+T細(xì)胞與DCs共培養(yǎng),用ELISA檢測培養(yǎng)上清液中IFN-γ水平�；旌狭馨图�(xì)胞反應(yīng)檢測各組DCs對CD4~+T淋巴細(xì)胞刺激增殖能力的影響。 4.下調(diào)DC-SIGN后對巨噬細(xì)胞活化吞噬殺滅結(jié)核桿菌的影響:用與DCs共培養(yǎng)后的初始CD4~+CD45RA~+T細(xì)胞與培養(yǎng)9天后的巨噬細(xì)胞混合培養(yǎng),48h后檢測TNF-α水平和NO水平。透射電鏡觀察巨噬細(xì)胞吞噬結(jié)核桿菌情況,裂解巨噬細(xì)胞并接種培養(yǎng),一周后計數(shù)。 結(jié)果:1.外周血單核細(xì)胞誘導(dǎo)分化的DCs在培養(yǎng)的第7天出現(xiàn)典型的形態(tài)學(xué)特征。流式細(xì)胞儀檢測ManLAM組表達(dá)CD83,CD86,DC-SIGN水平低于DCs組(p0.05)。ELISA檢測ManLAM組IL-12水平低于DCs組,IL-10水平高于DCs組(p0.05)。 2.①熒光顯微鏡下顯示各個轉(zhuǎn)染組的細(xì)胞表面出現(xiàn)綠色熒光,當(dāng)感染復(fù)數(shù)(MOI)為20時,DCs感染病毒的效率達(dá)85%。RT-PCR證實RNA干擾后DC-SIGN mRNA表達(dá)水平顯著低于陰性對照組和DCs組,差異有統(tǒng)計學(xué)意義(p0.05)。Western Blot檢測干擾組DCs內(nèi)總DC-SIGN水平顯著低于陰性對照組和DCs組,差異有統(tǒng)計學(xué)意義(p0.05)。②流式細(xì)胞儀檢測DC-SIGN陽性率在RNAi組、ManLAM組及ManLAM~+RNAi組與DCs組相比顯著下調(diào),差異有統(tǒng)計學(xué)意義(p0.05);各組間HLA-DR陽性率相比較差異無顯著性(p0.05)。ManLAM組CD83和CD86表達(dá)低于其余各組(p0.05);其余各組間相比差異無統(tǒng)計學(xué)意義(p0.05)。ELISA檢測顯示ManLAM組IL-10表達(dá)水平高于其他組(p0.05),ManLAM+RNAi組高于DCs組及RNAi組(p0.05),其余各組間差異無統(tǒng)計學(xué)意義(p0.05);ManLAM組IL-12表達(dá)水平低于其他各組(p0.05),其余各組差異無統(tǒng)計學(xué)意義(p0.05)。 3. ELISA檢測DCs與初始CD4~+ CD45RA~+T細(xì)胞共培養(yǎng)液中IFN-γ水平顯示,ManLAM組低于其余各組(p0.05);ManLAM+RNAi組IFN-γ水平低于DCs組(p0.05);其余各組間差異無統(tǒng)計學(xué)意義(p0.05)。混合淋巴細(xì)胞反應(yīng)檢測顯示ManLAM組CPM值低于其余各組(p0.05),其余各組間差異無統(tǒng)計學(xué)意義(p0.05)。 4. ELISA檢測T細(xì)胞與巨噬細(xì)胞共培養(yǎng)液中TNF-α水平顯示,ManLAM組TNF-α水平低于DCs組(p0.05),ManLAM+RNAi組TNF-α水平高于ManLAM組(p0.05);硝酸還原法檢測NO水平顯示,ManLAM組NO水平低于DCs組(p0.05),ManLAM+RNAi組NO水平高于ManLAM組(p0.05)。巨噬細(xì)胞內(nèi)活菌計數(shù)顯示,ManLAM組高于DCs組(p0.05),ManLAM+RNAi組活菌量低于ManLAM組(p0.05)。 結(jié)論: 1. ManLAM能干擾DCs成熟,下調(diào)DCs誘導(dǎo)的淋巴細(xì)胞增殖能力和激活初始CD4~+CD45RA~+T細(xì)胞向Th1細(xì)胞分化能力,降低巨噬細(xì)胞活化,最終導(dǎo)致巨噬細(xì)胞殺滅結(jié)核桿菌能力降低。 2.慢病毒介導(dǎo)的RNA干擾能有效的抑制DCs表面DC-SIGN分子表達(dá),DC-SIGN表達(dá)降低對DCs成熟度及DCs誘導(dǎo)的下游免疫功能無影響。 3.慢病毒介導(dǎo)的RNA干擾人DC-SIGN表達(dá)能恢復(fù)ManLAM對DCs成熟的抑制及DCs誘導(dǎo)的下游免疫功能,最終恢復(fù)或部分恢復(fù)下游巨噬細(xì)胞殺滅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.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392.12

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