人骨髓间充质干细胞体外扩增及其免疫调节作用的实验研究

发布时间：2018-05-09 05:10

本文选题：间充质干细胞 + 骨髓　；参考：《山东大学》2006年硕士论文

【摘要】：目的：研究骨髓间充质干细胞(Mesenchymal Stem Cells，MSCs)对PHA或异体抗原作用下的T淋巴细胞分泌功能的免疫调节作用，初步探讨MSCs诱导免疫耐受，降低移植物抗宿主病(graft-versus-host disease，GVHD)发生率的机制。研究方法：通过Ficoll分离法分离骨髓单个核细胞，体外培养扩增出MSCs，并通过形态学特征及流式细胞术检测其表面标志加以鉴定。通过Ficoll分离法和尼龙棉柱法获取外周血T淋巴细胞，将不同数量的MSCs与PHA激活的T细胞共培养4天，，收集培养上清液；将不同数量的MSCs加入混合淋巴细胞反应(mixed lymphocyte reaction，MLR)体系共培养7天，收集培养上清液；将不同浓度的MSCs培养上清加入MLR体系共培养7天，收集培养上清液。应用酶联免疫吸附试验(enzyme-linked immunosorbent assay，ELISA)分别检测各培养上清液中T细胞分泌的细胞因子水平，包括IL-2和INF-γ(主要由Th1细胞分泌)，IL-4和IL-10(主要由Th2细胞分泌)。结果：骨髓单个核细胞接种于培养瓶后，2～4天可见成纤维样贴壁细胞逐渐出现，增殖速度较慢；7～11天散在的成纤维样贴壁细胞增殖较快，细胞形态均一；2周左右，细胞生长呈漩涡状或平行状排列，细胞融合可到达90％。传代细胞生长迅速，主要呈成纤维样，约第7～10天可达到融合。流式细胞仪检测人骨髓MSCs表面抗原高表达CD29、CD105、CD166；而低表达CD34、CD45、HLA-DR。
[Abstract]:Objective: To study the immunoregulation effect of Mesenchymal Stem Cells (MSCs) on the secretory function of T lymphocytes under the action of PHA or allogenic antigen, and to explore the mechanism of MSCs induction of immune tolerance and the reduction of the incidence of graft versus host disease (graft-versus-host disease, GVHD).
Methods: Ficoll separation method was used to separate bone marrow mononuclear cells. MSCs was cultured and amplified in vitro. The morphological characteristics and flow cytometry were used to identify the surface markers. T lymphocytes were obtained by Ficoll separation and nylon cotton column method, and the number of MSCs and PHA activated T cells were collected for 4 days. The supernatant was cultured, and different amounts of MSCs were added to the mixed lymphocyte reaction (mixed
Lymphocyte reaction, MLR) system was cultured for 7 days, and the supernatant was collected and cultured. The culture supernatant of different concentration of MSCs was added to MLR system for 7 days to collect culture supernatant. The level of cytokines secreted by T cells in each culture supernatant was detected by enzyme linked immunosorbent assay (enzyme-linked immunosorbent assay, ELISA), including I. L-2 and INF- gamma (mainly secreted by Th1 cells), IL-4 and IL-10 (mainly secreted by Th2 cells).
Results: after the bone marrow mononuclear cells were inoculated in the culture bottle, the fibroid adherent cells appeared gradually and the proliferation rate was slow on the 2~4 day, and the proliferation of fibroid adherent cells on the 7~11 day was faster and the cell morphology was uniform, and the cell growth was whirlpool or parallel in 2 weeks, and the cell fusion could reach the growth of the 90%. passages cells. Rapid, mainly fibrous like, about seventh to 10 days to achieve fusion. Flow cytometry detection of human bone marrow MSCs surface antigen high expression of CD29, CD105, CD166, and low expression of CD34, CD45, HLA-DR.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R329.2

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