本文關(guān)鍵詞： 骨髓間充質(zhì)干細(xì)胞 細(xì)胞培養(yǎng) 流式細(xì)胞術(shù) B淋巴細(xì)胞 增殖 免疫球蛋白 凋亡 免疫調(diào)節(jié)　出處：《蘭州大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的探討骨髓間充質(zhì)干細(xì)胞(bone marrow mesenchymal stem cells,MSCs)在體外對異體外周血B淋巴細(xì)胞的免疫調(diào)節(jié)作用。 方法密度梯度離心法從骨髓中分離單個核細(xì)胞,加入含10%胎牛血清的LG-DMEM培養(yǎng)液,將細(xì)胞的密度調(diào)整到2×10~5個/ml,接種到培養(yǎng)瓶中,48 h后更換新鮮培養(yǎng)液,以后每2～3 d換液1次。當(dāng)細(xì)胞鋪滿培養(yǎng)瓶底90%以上時,按常規(guī)方法進(jìn)行細(xì)胞傳代培養(yǎng)。用流式細(xì)胞術(shù)檢測培養(yǎng)細(xì)胞的CD3、CD4、CD8、CD13、CD22、CD33、CD34、CD44、HLA-DR表達(dá)情況。常規(guī)從外周血中分離單個核細(xì)胞,L-亮氨酸甲酯去除單核細(xì)胞,以2-氨乙基硫脲溴化物(AET)處理的綿羊紅細(xì)胞(SRBC)花環(huán)形成法,去除T淋巴細(xì)胞獲得純化的B淋巴細(xì)胞。用羊抗人IgM單克隆抗體(Anti-IgM)刺激與或未與MSCs及其培養(yǎng)上清共培養(yǎng)3天的B淋巴細(xì)胞,應(yīng)用MTT法測B淋巴細(xì)胞的增殖,ELISA法測培養(yǎng)上清中免疫球蛋白IgG、IgM的產(chǎn)生,應(yīng)用流式細(xì)胞儀分別檢測與MSCs共培養(yǎng)24h、48h后B淋巴細(xì)胞的凋亡。 結(jié)果實(shí)驗(yàn)中觀察到,梯度離心所得單個核細(xì)胞接種2～3h細(xì)胞開始貼壁,24h后貼壁細(xì)胞數(shù)目不再增加,1～2w后開始迅速增殖,細(xì)胞多為梭形,培養(yǎng)至3w周左右貼壁細(xì)胞接近80%～90%融合。傳代培養(yǎng)后,傳代細(xì)胞在12～24 h內(nèi)全貼壁,生長迅速,形態(tài)均一,大約7d左右達(dá)到完全融合。傳代擴(kuò)增細(xì)胞以流式細(xì)胞術(shù)檢測,結(jié)果顯示CD3、CD4、CD8、CD22、CD33、CD34和HLA-DR均表達(dá)陰性,高表達(dá)CD13,CD44。 在體外活化B淋巴細(xì)胞反應(yīng)體系中加入異體MSCs,顯示MSCs及其培養(yǎng)上清抑制由絲裂原Anti-IgM誘導(dǎo)的B淋巴細(xì)胞的增殖,且這種抑制程度和MSCs的細(xì)胞數(shù)量及其培養(yǎng)上清濃度有關(guān)。體外與不同比例MSCs共培養(yǎng)的B淋巴細(xì)胞,MSCsⅡ(B∶MSCs10∶1)、MSCsⅢ(B∶MSCs 1∶1)組A值明顯低于未加MSCs的對照組A值(均P＜0.01),并且MSCsⅢ組A值顯著低于MSCsⅠ(B∶MSCs 50∶1)組(P＜0.05)。用含有12.5%、25%、50%不同MSCs培養(yǎng)上清液濃度的培養(yǎng)基培養(yǎng)B淋巴細(xì)胞3d后,其中50%組A值同對照組相比有統(tǒng)計(jì)學(xué)意義(P＜0.05),并且顯著低于12.5%組的A值(P＜0.05)。ELISA檢測顯示MSCs及其培養(yǎng)上清液抑制B淋巴細(xì)胞分泌免疫球蛋白,并且隨著MSCs數(shù)量的及其上清濃度的增加,這種抑制也越明顯。與B淋巴細(xì)胞加刺激物組相比,B∶MSCs 1∶1組及50%super組分泌的IgM明顯減少(P＜0.01),而只有B∶MSCs 1∶1組分泌的IgG同對照組相比差異有統(tǒng)計(jì)學(xué)意義(P＜0.05)。 MSCs不誘導(dǎo)B淋巴細(xì)胞的凋亡;B淋巴細(xì)胞與MSCs共培養(yǎng)24h、48h,加或不加Anti-IgM,各組間細(xì)胞凋亡率組比較差異無統(tǒng)計(jì)學(xué)意義(P＞0.05)。MSCs對B淋巴細(xì)胞的抑制具有可逆性。收集和MSCs共培養(yǎng)3d的B淋巴細(xì)胞,重新用Anti-IgM刺激增殖,與未加Anti-IgM的共孵B淋巴細(xì)胞組A值相比,二者間差異有統(tǒng)計(jì)學(xué)意義(P＜0.0)。 結(jié)論MSCs對異體外周血B淋巴細(xì)胞存在免疫調(diào)節(jié)作用,并且這種調(diào)控機(jī)制是復(fù)雜的,不僅與MSCs細(xì)胞數(shù)量有關(guān),還與細(xì)胞間的相互作用和MSCs分泌的細(xì)胞因子有關(guān)。
[Abstract]:Objective to investigate the bone marrow mesenchymal stem cells of bone marrow mesenchymal stem cells. The immunomodulatory effect of MSCs on peripheral blood B lymphocytes in vitro. Methods Mononuclear cells were isolated from bone marrow by density gradient centrifugation, and LG-DMEM medium containing 10% fetal bovine serum was added to adjust the cell density to 2 脳 10 ~ 5 / ml. The fresh culture medium was changed after 48 h of inoculation in the culture bottle, and then changed every 2 ~ 3 days. When the cells were covered with the culture bottle bottom 90% or more. The cell culture was carried out by conventional method. The CD3T CD4, CD8, CD13, CD22, CD32, CD34 and CD44 of the cultured cells were detected by flow cytometry (FCM). HLA-DR expression. Mononuclear cells were routinely isolated from peripheral blood to remove monocytes from L- leucine methyl ester. The rosette formation of sheep red blood cell (SRBC) treated with 2-aminoethyl thiourea bromide was studied. Purified B lymphocytes were obtained by removing T lymphocytes. B lymphocytes were stimulated with or not co-cultured with MSCs and its supernatant for 3 days with sheep anti-human IgM monoclonal antibody Anti-IgM. MTT method was used to measure the proliferation of B lymphocytes and Elisa was used to detect the immunoglobulin IgM production in the supernatant. Flow cytometry was used to detect MSCs co-culture for 24 hours. Apoptosis of B lymphocytes was observed after 48 hours. Results it was observed that the mononuclear cells inoculated with gradient centrifugation for 24 hours after inoculation for 2 hours began to adhere to the wall, and the number of adherent cells did not increase after 2 weeks, and the cells began to proliferate rapidly, and most of the cells were fusiform. After 3 weeks of culture, the adherent cells were close to 80% and 90% fused. After subculture, the cells were adherent to the wall completely within 1224 h. The cells grew rapidly and the morphology was uniform. After about 7 days, the cells were fused completely. The results of flow cytometry showed that CD3 + CD4 + CD8 + CD22 + CD33 was detected by flow cytometry. Both CD34 and HLA-DR were negative and CD13 CD44 was highly expressed. MSCs and its culture supernatant inhibited the proliferation of B lymphocytes induced by mitogen Anti-IgM. The inhibition degree was related to the number of MSCs cells and the concentration of supernatant. The A value of MSCs 鈪，

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