骨髓间充质干细胞调控Aβ激活的小胶质细胞活性及其可能机制

发布时间：2018-05-26 08:21

本文选题：CD200-CD200R + 小胶质细胞　；参考：《苏州大学》2014年硕士论文

【摘要】：目的：小胶质细胞（microglia，MI）是脑内的免疫细胞，其功能状态与微环境相关。CD200-CD200R途径被认为是维持小胶质细胞静息状态的重要机制之一。在老龄脑中，神经元跨膜糖蛋白CD200表达减少，由于CD200-CD200R途径免疫抑制信号的减弱，在β-淀粉样蛋白（amyloid β-peptide，Aβ）异常聚集时，MI易于过度激活而产生多种炎性因子、趋化因子，产生慢性炎症反应，促进神经元的丢失。骨髓间充质干细胞(Bone Mesenchymal Stem Cells, BMSCs)是具有多向分化潜能的干细胞，移植BMSCs是阿尔茨海默病的潜在治疗途径之一。CD200被证实表达在骨髓间充质干细胞膜上。由此设想，抑制性免疫调控分子CD200是BMSCs治疗AD的作用机制之一。本实验通过原代培养得到人骨髓间充质干细胞和小胶质细胞，观察BMSCs上的CD200及MI的CD200R表达，以及加入抗CD200抗体能否逆转BMSCs对Aβ激活的MI的抑制，以期探讨BMSCs对于体外Aβ作用的小胶质细胞活性调控可能的新机制。方法：原代分离培养、纯化小胶质细胞、骨髓间充质干细胞，，观察其形态，免疫荧光鉴定BMSCs上CD200、MI上CD200R的表达，用Aβ激活小胶质细胞，进行BMSCs和MI的体外共培养，分为MI组、BMSCs组、MI+Aβ组、MI+BMSCs组、MI+Aβ+BMSCs组、MI+Aβ+BMSCs+抗CD200抗体组，用ELISA法检测各组培养48小时后上清液中炎性因子肿瘤坏死因子-α（Tumor Necrosis Factor-α，TNF-α）、白介素-6（interleukin-6，IL-6）、一氧化氮（Nitric oxide，NO）含量。所有统计分析均用SPSS16.0统计软件分析，P0.05表示差异具有统计学意义。结果：培养的MI表达CD200R，BMSCs表达CD200。Aβ作用后的小胶质细胞呈阿米巴样，产生的TNF-α、IL-6、NO量较静息状态时增加（P0.05），说明体外Aβ作用后小胶质细胞活化，BMSCs与活化的MI组共培养后TNF-α、IL-6、NO减少，差别有统计学意义（p0.05）,加入抗CD200抗体组后小胶质细胞活化形态、炎性因子含量有所改变，较Aβ活化的MI组低，但较BMSCs和活化的MI组高，差别两两比较有统计学意义（p0.05）。结论：BMSCs可通过CD200-CD200R接触抑制体外培养的Aβ激活的小胶质细胞的过度活化。
[Abstract]:Aim: microglia Mi) is an immune cell in the brain. Its functional state is related to microenvironment. CD200-CD200R pathway is considered to be one of the important mechanisms to maintain the resting state of microglia cells. In the aged brain, the expression of transmembrane glycoprotein (CD200) in neurons decreased. Due to the decrease of CD200-CD200R pathway immunosuppressive signal, MI was prone to over-activate during the abnormal aggregation of amyloid 尾 -peptideA 尾 in 尾 -amyloid, resulting in a variety of inflammatory factors, chemokines, and chemokines. Chronic inflammatory response is produced to promote the loss of neurons. Bone marrow mesenchymal stem cells (BMSCs) are stem cells with multiple differentiation potential. Transplantation of BMSCs is one of the potential therapeutic pathways for Alzheimer's disease. CD200 has been confirmed to be expressed on the membrane of bone marrow mesenchymal stem cells (BMSCs). It is assumed that the inhibitory immunomodulatory molecule CD200 is one of the mechanisms of BMSCs in the treatment of AD. In this study, human bone marrow mesenchymal stem cells and microglial cells were obtained by primary culture to observe the CD200R expression of CD200 and MI on BMSCs, and whether the addition of anti-CD200 antibody could reverse the inhibition of A 尾 -activated MI induced by BMSCs. The aim of this study was to explore the possible mechanism of microglial activity regulation by BMSCs on A 尾 in vitro. Methods: microglial cells and bone marrow mesenchymal stem cells were isolated and cultured in primary culture, and their morphology was observed. The expression of CD200R on CD200mMI on BMSCs was identified by immunofluorescence. Microglia were activated by A 尾 and co-cultured with BMSCs and MI in vitro. BMSCs group was divided into MI A 尾 group and MI A 尾 BMSCs group. The contents of TNF- 伪 Tumor Necrosis Factor- 伪 TNF- 伪, interleukin-6 interleukin-6IL-6, nitric oxide (no) in the supernatant of each group were detected by ELISA method. All the statistical analyses were analyzed by SPSS16.0 statistical software. The difference was statistically significant (P 0.05). Results: the expression of CD200.A 尾 in microglial cells of cultured MI expressed CD200R- BMSCs was amoeba like, and the amount of TNF- 伪 IL-6 no produced was higher than that of P0.05in resting state, which indicated that the level of TNF- 伪 IL-6no decreased after microglial activation of BMSCs and activated MI co-cultured in vitro. The difference was statistically significant (P 0.05). The activation morphology of microglia and the content of inflammatory factors were changed after adding anti CD200 antibody, which was lower than that in MI group activated by A 尾, but higher than that in BMSCs group and activated MI group. The difference was statistically significant compared with that of BMSCs group and activated MI group (P 0.05). ConclusionBMSCs can inhibit the overexpression of A 尾 -activated microglia by CD200-CD200R contact.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R749.16

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