本文選題：樹突狀細(xì)胞 + 調(diào)節(jié)性　； 參考：《山西醫(yī)科大學(xué)》2012年碩士論文

【摘要】：目的： 體外誘導(dǎo)小鼠骨髓來源調(diào)節(jié)性樹突狀細(xì)胞的生成，檢測IL-10、SOCS1、TLR4及NF-κB基因在其中的表達(dá)，探討其發(fā)揮免疫作用的機(jī)制。 方法： 1、分離、提純小鼠骨髓來源樹突狀細(xì)胞，分為四組進(jìn)行體外培養(yǎng)，分別標(biāo)記為A、B、C、D。 2、培養(yǎng)過程中A、B組培養(yǎng)液中加入GM-CSF+IL-4，C、D組培養(yǎng)液中加入GM-CSF+IL-10+TGF-β1，培養(yǎng)第3天，棄去上清，去除懸浮細(xì)胞，加入新鮮培養(yǎng)液，補(bǔ)充細(xì)胞因子，之后隔天半量換液，至第8天，B、D組給予LPS刺激48h，促進(jìn)細(xì)胞進(jìn)一步分化。 3、用倒置顯微鏡觀察細(xì)胞生長情況，記錄生長變化。 4、收集第10天的各組細(xì)胞及上清液，通過流式細(xì)胞儀，ELISA以及RT-PCR的方法對細(xì)胞進(jìn)行鑒定和檢測。 結(jié)果： 1、 GM-CSF、IL-4、IL-10、TGF-β1、LPS等因子進(jìn)行體外培養(yǎng)小鼠骨髓來源細(xì)胞能誘導(dǎo)出具有樹突狀結(jié)構(gòu)的細(xì)胞，，且加入LPS組即B、D組樹突狀突起更加典型，B組分叉更多，突起粗壯。 2、流式細(xì)胞儀檢測示B組表達(dá)更高水平的CD11c、MHC-Ⅱ、CD80、CD86，分別為(94.181.50)%，(81.172.45)%，(72.852.24)%，(24.781.57)%，呈現(xiàn)成熟樹突狀細(xì)胞（mDC）的特性，而A、C、D組則低表達(dá)的上述因子，分別為未成熟樹突狀細(xì)胞（iDC）、調(diào)節(jié)性樹突狀細(xì)胞前體（pDCreg）及調(diào)節(jié)性樹突狀細(xì)胞（DCreg）。 3、 ELISA檢測各組細(xì)胞上清液中IL-12的表達(dá)水平，四組結(jié)果依次為12.281.67，35.302.36，10.322.23，11.172.82，發(fā)現(xiàn)B組表達(dá)明顯高于其他各組(P0.05)，差異有統(tǒng)計學(xué)意義，其余三組間無明顯差異。 4、實時熒光定量PCR結(jié)果顯示：與iDC，mDC相比，IL-10和SOCS1在pDCreg，DCreg中高表達(dá)（P0.05），差異有統(tǒng)計學(xué)意義；而TLR4和NF-κB在mDC中高表達(dá)，其他各組均低表達(dá)（P0.05），差異有統(tǒng)計學(xué)意義，其余三組間無明顯差異。 結(jié)論： 體外成功誘導(dǎo)出骨髓來源調(diào)節(jié)性樹突狀細(xì)胞，發(fā)現(xiàn)IL-10，SOCS1基因表達(dá)在調(diào)節(jié)性樹突狀細(xì)胞形成過程中發(fā)揮重要作用，為臨床免疫性疾病的治療開辟新的途徑。
[Abstract]:Aim: to induce the formation of mouse bone marrow-derived regulatory dendritic cells in vitro and detect the expression of TLR4 and NF- 魏 B genes in mouse bone marrow-derived dendritic cells. Methods: 1. Mouse bone marrow derived dendritic cells were isolated and purified and divided into four groups for culture in vitro. GM-CSF IL-10 TGF- 尾 1 was added to GM-CSF IL-4CU D culture medium during the culture process, and the supernatant was discarded, suspended cells were removed, fresh culture medium was added and cytokine was added to the culture medium, and GM-CSF IL-10 TGF- 尾 1 was added to the culture medium. On the 8th day, group D was stimulated with LPS for 48 h to promote cell differentiation. 3. The growth of cells was observed by inverted microscope. The changes of growth were recorded. 4. The cells and supernatants of each group were collected on the 10th day. The cells were identified and detected by flow cytometry (FCM) Elisa and reverse transcription-polymerase chain reaction (RT-PCR). Results: 1. GM-CSF IL-4 IL-10 TGF- 尾 1 LPS could induce dendritic cells from mouse bone marrow in vitro, and the dendritic processes in group B were more typical than those in group B, and the dendritic processes in group B were more typical than those in group B. 2FCM analysis showed that group B expressed a higher level of CD11cCHC- 鈪

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