小鼠骨髓来源调节性树突状细胞的体外培养和鉴定

发布时间：2018-06-28 01:08

  本文选题：树突状细胞 + 调节性　； 参考：《山西医科大学》2012年硕士论文

【摘要】：目的： 体外诱导小鼠骨髓来源调节性树突状细胞的生成，检测IL-10、SOCS1、TLR4及NF-κB基因在其中的表达，探讨其发挥免疫作用的机制。 方法： 1、分离、提纯小鼠骨髓来源树突状细胞，分为四组进行体外培养，分别标记为A、B、C、D。 2、培养过程中A、B组培养液中加入GM-CSF+IL-4，C、D组培养液中加入GM-CSF+IL-10+TGF-β1，培养第3天，弃去上清，去除悬浮细胞，加入新鲜培养液，补充细胞因子，之后隔天半量换液，至第8天，B、D组给予LPS刺激48h，促进细胞进一步分化。 3、用倒置显微镜观察细胞生长情况，记录生长变化。 4、收集第10天的各组细胞及上清液，通过流式细胞仪，ELISA以及RT-PCR的方法对细胞进行鉴定和检测。 结果： 1、 GM-CSF、IL-4、IL-10、TGF-β1、LPS等因子进行体外培养小鼠骨髓来源细胞能诱导出具有树突状结构的细胞，，且加入LPS组即B、D组树突状突起更加典型，B组分叉更多，突起粗壮。 2、流式细胞仪检测示B组表达更高水平的CD11c、MHC-Ⅱ、CD80、CD86，分别为(94.181.50)%，(81.172.45)%，(72.852.24)%，(24.781.57)%，呈现成熟树突状细胞（mDC）的特性，而A、C、D组则低表达的上述因子，分别为未成熟树突状细胞（iDC）、调节性树突状细胞前体（pDCreg）及调节性树突状细胞（DCreg）。 3、 ELISA检测各组细胞上清液中IL-12的表达水平，四组结果依次为12.281.67，35.302.36，10.322.23，11.172.82，发现B组表达明显高于其他各组(P0.05)，差异有统计学意义，其余三组间无明显差异。 4、实时荧光定量PCR结果显示：与iDC，mDC相比，IL-10和SOCS1在pDCreg，DCreg中高表达（P0.05），差异有统计学意义；而TLR4和NF-κB在mDC中高表达，其他各组均低表达（P0.05），差异有统计学意义，其余三组间无明显差异。 结论： 体外成功诱导出骨髓来源调节性树突状细胞，发现IL-10，SOCS1基因表达在调节性树突状细胞形成过程中发挥重要作用，为临床免疫性疾病的治疗开辟新的途径。
[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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