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  本文選題：樹突狀細(xì)胞 + CD11c��； 參考：《重慶醫(yī)科大學(xué)》2010年碩士論文

【摘要】： 目的:以小鼠骨髓細(xì)胞為前體,建立一種高效、簡便的體外擴(kuò)增、分離培養(yǎng)未成熟樹突狀細(xì)胞(imDC)的方法,從而為imDC的實驗研究和臨床應(yīng)用奠定基礎(chǔ)。 方法:實驗分為GM/IL-5、GM/IL-7和GM/IL-LPS三組:制備C57BL/6小鼠骨髓細(xì)胞,三羥甲基氨基甲烷-氯化銨緩沖液(Tris-NH4Cl)裂解紅細(xì)胞后,以重組小鼠粒細(xì)胞巨噬細(xì)胞集落刺激因子(rmGM-CSF)和重組小鼠白細(xì)胞介素-4(rmIL-4)對其聯(lián)合誘導(dǎo)培養(yǎng),48 h后通過貼壁法去除懸浮的粒細(xì)胞及淋巴細(xì)胞,之后隔天半量換液一次,最后根據(jù)DC培養(yǎng)72 h后自動從塑料板分離的特性分別于第五天(GM/IL-5組)、第七天(GM/IL-7組)收集懸浮及疏松貼壁細(xì)胞,而GM/IL-LPS組則是于第六天半量換液后添加大劑量脂多糖(LPS)繼續(xù)培養(yǎng)18 h后收集所得;將收獲的三組細(xì)胞進(jìn)行形態(tài)及功能學(xué)的鑒定,即:掃描電鏡觀察細(xì)胞形態(tài)、流式細(xì)胞術(shù)(FCM)測定細(xì)胞表面分子的表達(dá)、同種異體混合淋巴細(xì)胞反應(yīng)(MLR)檢測三組DC刺激同種異體T細(xì)胞增殖的能力。 結(jié)果:掃描電鏡下所收集的三組細(xì)胞均具有典型DC形態(tài);流式結(jié)果表明細(xì)胞表面均高表達(dá)小鼠髓源性DC相對特異性標(biāo)志CD11c,表達(dá)率達(dá)73%以上,GM/IL-5組DC細(xì)胞表面CD40、CD86、MHC-Ⅱ的表達(dá)率分別為34.46%、34.21%、45.16%,GM/IL-7組則分別為46.99%、68.79%、94.37%,GM/IL-LPS組為78.68%、89.84%、96.75%;MLR中GM/IL-5組DC刺激同種異體T細(xì)胞活化增殖的能力不如GM/IL-7、GM/IL-LPS組強(qiáng)(P0.05)。 結(jié)論:此種方法能于體外定向誘導(dǎo)和擴(kuò)增出大量具有典型DC形態(tài)、細(xì)胞表型及較高純度的髓源性imDC,不僅具有降低實驗成本、縮短培養(yǎng)時間等優(yōu)點,而且能有效界定未成熟與成熟DC間的分化時限,這將為后續(xù)研究imDC在器官移植后誘導(dǎo)機(jī)體免疫耐受的臨床應(yīng)用奠定基礎(chǔ)。
[Abstract]:Objective: to establish an efficient and simple method for isolation and culture of immature dendritic cells from mouse bone marrow cells in vitro, so as to lay a foundation for the experimental study and clinical application of imDC.Methods: the experiment was divided into three groups: C57BL/6 mice bone marrow cells were prepared by GM- / IL-5 GM- / IL-7 and GM/IL-LPS. The erythrocytes were lysed with trihydroxymethylaminomethane-ammonium chloride buffer solution (Tris-NH _ 4Cl).Recombinant mouse granulocyte macrophage colony-stimulating factor (rmGM-CSF) and recombinant mouse interleukin-4rmIL-4 (rmIL-4) were co-induced to remove suspended granulocytes and lymphocytes by adherent method for 48 h.Finally, suspension and loose adherent cells were collected according to the characteristics of DC cultured for 72 hours from plastic plates on the fifth day (GM- / IL-5 group) and on the 7th day (GM- / IL-7 group).In the GM/IL-LPS group, the cells were cultured for 18 hours after six and a half days of liquid exchange, and the three groups of cells were identified by morphology and function, that is, the morphology of the cells was observed by scanning electron microscope (SEM), and the cell morphology was observed by scanning electron microscope (SEM).Flow cytometry (FCM) was used to detect the expression of cell surface molecules, and MLR was used to detect the ability of DC to stimulate the proliferation of allogeneic T cells.Results: the three groups of cells collected under scanning electron microscope had typical DC morphology.The ability of T cells to activate and proliferate was not as strong as that of GM- / IL-7 / GM- / IL-LPS group.Conclusion: this method can induce and amplify a large number of myelogenous imDCs with typical DC morphology, cell phenotype and high purity in vitro, which can not only reduce the experimental cost, but also shorten the culture time.Moreover, it can effectively define the differentiation time between immature and mature DC, which will lay a foundation for further study on the clinical application of imDC in inducing immune tolerance after organ transplantation.
【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

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本文編號：1731561