本文選題：樹突狀細(xì)胞 + 阻斷療法��； 參考：《石河子大學(xué)》2008年碩士論文

【摘要】： 目的:依據(jù)GVHD發(fā)生機(jī)制構(gòu)建體外DC誘導(dǎo)T淋巴細(xì)胞的調(diào)節(jié)模型,觀察供者未成熟樹突狀細(xì)胞(immature dendritic cell,imDC)刺激白體T細(xì)胞增殖的能力,探討利用imDC治移植物抗宿主病(GVHD)臨床應(yīng)用的可行性。 方法:從健康供者外周血分離單核細(xì)胞,采用重組人粒細(xì)胞巨噬細(xì)胞集落刺激因子(rhGM-CSF)和白細(xì)胞介素(IL)-4聯(lián)合培養(yǎng)5天,誘導(dǎo)其分化成imDC;誘導(dǎo)培養(yǎng)7天,加脂多糖(LPS)刺激18小時(shí)后,分化成熟樹突狀細(xì)胞(mature dendritic cell,mDC)。并通過倒置顯微鏡和HE染色觀察細(xì)胞形態(tài),細(xì)胞滴片免疫組化檢測mDC表面MHC-II類分子HLA-DR、CD86的表達(dá)情況.利用流式細(xì)胞儀檢測imDC和mDC表面DC特異性分子標(biāo)記CDla和成熟標(biāo)記CD83的陽性表達(dá)率。采用單向MLR方法,進(jìn)行供受者混合淋巴細(xì)胞培養(yǎng),構(gòu)建DC誘導(dǎo)T淋巴細(xì)胞的調(diào)節(jié)模型,將實(shí)驗(yàn)設(shè)為3組:對照組、imDC組和mDC組,共孵育48小時(shí)后,加入CCK-8,用酶標(biāo)儀檢測各組OD值,比較供者imDC和mDC刺激自體T細(xì)胞增殖的能力。 結(jié)果:(1)培養(yǎng)5天后細(xì)胞呈半懸浮狀生長,周邊毛刺較粗短,形態(tài)不一,細(xì)胞體積較單核細(xì)胞增大具有典型的imDC形態(tài)學(xué)特征,流式細(xì)胞儀檢測CDla、CD83和雙抗分別表達(dá)為(67.06±0.93)%、(66.82±5.06)%和(62.34±1.94)%;培養(yǎng)8天后細(xì)胞呈單個(gè)懸浮狀生長,胞體圓形,體積較大,細(xì)胞周邊可見明顯較長突起,具有典型mDC形態(tài)學(xué)特征,細(xì)胞滴片免疫組化結(jié)果顯示HLA-DR、CD86陽性,流式細(xì)胞儀檢測CDla、CD83和雙抗表達(dá)分別為(64.98±2.99)%、(86.44±4.10)%和(65.16±0.55)%。(2)單向MLR法混臺(tái)淋巴細(xì)胞培養(yǎng)共孵育48小時(shí)后,加入CCK-8檢測OD值,imDC組與對照組比較無統(tǒng)計(jì)學(xué)意義(P0.05):mDC組與對照組、imDC組比較均有顯著統(tǒng)計(jì)學(xué)意義(P0.01),對自體T細(xì)胞的刺激指數(shù)大于2.00(SI=2.22),與2.00相比差異有統(tǒng)計(jì)學(xué)意義(P0.05)。 結(jié)論:形態(tài)學(xué)觀察、免疫組化及流式細(xì)胞儀細(xì)胞表型檢測結(jié)果提示,rhGM-CSF+IL-4聯(lián)合成功誘導(dǎo)培養(yǎng)出了imDC和mDC;在單向MLR法構(gòu)建的DC誘導(dǎo)T淋巴細(xì)胞的調(diào)節(jié)模型中CCK-8法檢測結(jié)果提示imDC可以誘導(dǎo)白體T細(xì)胞低應(yīng)答,有可能成為防治GVHD的一種方法。
[Abstract]:Aim: to establish a regulatory model of T lymphocytes induced by DC in vitro according to the mechanism of GVHD, and to observe the ability of donor immature dendritic cells (immature dendritic cells) to stimulate the proliferation of leukocytes. To explore the feasibility of clinical application of IMDC in the treatment of graft-versus host disease (GVHD). Methods: mononuclear cells were isolated from the peripheral blood of healthy donors and co-cultured with recombinant human granulocyte macrophage colony stimulating factor (rhGM-CSF) and interleukin-4 (IL-4) for 5 days to induce their differentiation into imDC.After 7 days of induction and 18 hours of lipopolysaccharide (LPS) stimulation, Differentiation of mature dendritic cells (mature dendritic cell / mDC). The cell morphology was observed by inverted microscope and HE staining. The expression of MHC-II molecule HLA-DRN CD86 on MDC was detected by immunohistochemistry. The positive rates of DC specific molecular marker CDla and mature marker CD83 on the surface of imDC and mDC were detected by flow cytometry. Single MLR method was used to culture donor mixed lymphocytes, and the regulatory model of T lymphocytes induced by DC was established. The experiment was divided into three groups: control group and mDC group. After incubation for 48 hours, CCK-8 was added to each group. OD value of each group was detected by enzyme marker. To compare the ability of donor imDC and mDC to stimulate the proliferation of autologous T cells. Results: (1) after 5 days of culture, the cells grew semi-suspended, the peripheral burr was shorter and thicker, the morphology was different, and the cell volume was larger than that of monocytes, which had typical morphological characteristics of imDC. The expression of CD83 and double antibody were (67.06 鹵0.93), (66.82 鹵5.06)% and (62.34 鹵1.94), respectively. The expression of CD83 and double antibody were (64.98 鹵2.99)%, (86.44 鹵4.10)% and (65.16 鹵0.55)%, respectively. (2) the expression of CD83 and double antibody were (64.98 鹵2.99), (86.44 鹵4.10)% and (65.16 鹵0.55), respectively. There was no significant difference in OD value between the control group and the control group (P0.05). The stimulation index of autologous T cells was more than 2.00 (SI-2.22) in the control group (P0.01), and the difference was significant compared with the control group (P0.05) (P0.05). Conclusion: morphological observation, The results of immunohistochemistry and flow cytometry showed that rhGM-CSF IL-4 combined with rhGM-CSF IL-4 could induce imDC and mDC.The CCK-8 method showed that imDC could induce T lymphocytes in the model of DC induced by unidirectional MLR. White body T cell low response, It may be a method to prevent GVHD.
【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392

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