人脐带间充质干细胞微泡对类风湿关节炎患者外周血淋巴细胞及炎性因子的影响

发布时间：2018-06-17 17:24

本文选题：类风湿关节炎 + 间充质干细胞微泡　；参考：《山西医科大学》2017年硕士论文

【摘要】：背景:类风湿关节炎(Rheumatoid Arthritis,RA)是一种慢性、系统性、炎症性自身免疫性疾病,其病理改变是机体免疫功能紊乱导致的滑膜增生、血管翳形成、骨及软骨组织的破坏。有研究的表明,Treg/Th17细胞的平衡失调对RA的发生、发展起着至关重要的作用[1,2]。间充质干细胞(Mesenchyma Stem Cells,MSCs)已被证实是一种具有高度增值能力和多向分化潜能的细胞[3],可通过调节炎症细胞及因子的表达,达到治疗RA的目的。近几年研究发现,间充质干细胞来源的微泡(Mesenchymal Stem Cells derived Microvesicles,MSC-MVs)同样具有免疫调节作用[5],同时可避免细胞治疗引起的血管栓塞、异位成骨等风险,因此逐渐成为新型的研究热点。目的:通过体外培养人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUC-MSCs)及分离人脐带间充质干细胞来源的微泡(human umbilical cord mesenchymal stem cells derived microvesicles,hUCMSC-MVs),将hUCMSC-MVs与RA患者外周血单个核细胞(Peripheral Blood Mononuclear Cells,PBMCs)体外共培养,观察hUCMSC-MVs对RA患者PBMC中Th17细胞、Treg细胞及炎性因子IL-17A、IL-1a、IL-2、TNF-α、TGF-β、IL-10表达的影响,探讨hUCMSC-MVs治疗RA是否有效,作用强度如何,是否与其浓度相关,进而从炎症细胞及因子途径阐述hUCMSC-MVs治疗RA可能的作用机制。方法:1.hUC-MSCs和hUCMSC-MVs的分离、鉴定(1)huc-mscs的分离、培养和鉴定:采用组织块培养方法体外分离、培养huc-mscs,倒置显微镜下观察其形态,流式细胞术检测表面抗原cd29、cd44、cd105、cd73、cd90、cd166、cd34、cd45,并在特定条件下对huc-mscs的成骨、成脂分化潜能进行鉴定;(2)hucmsc-mvs的分离和鉴定:huc-mscs“饥饿”培养24小时,使用差速离心提取上清液中的微泡,使用透射电镜观察其形态,并采用bca方法在酶标仪上测定其蛋白质含量。2.hucmsc-mvs与ra患者pbmcs共培养,观察其对th17细胞、treg细胞及相关炎性因子表达水平的影响(1)实验分组:共5组,分别是空白对照组、huc-mscs组(2×106个)、hucmsc-mvs组(30μg)、hucmsc-mvs组(60μg)、hucmsc-mvs组(90μg)。(2)检测指标:⑴流式细胞术测定th17细胞、treg细胞表达水平。⑵流式高通量多因子检测技术测定培养上清中促炎因子il-17a、il-1a、il-2、tnf-α和抗炎因子tgf-β、il-10的表达水平,收集数据做统计分析。结果:1.huc-mscs和hucmsc-mvs的分离、鉴定(1)huc-mscs的分离、培养和鉴定:倒置显微镜下观察细胞排列较整齐,呈较为均一的长梭形,类似成纤维细胞样外观。经流式细胞术检测,细胞表面cd29、cd44、cd73、cd90、cd105和cd166呈阳性表达,cd34、cd45成阴性表达。huc-mscs经成骨诱导分化,钙钴法染色成阳性;经成脂诱导分化,油红o染色成阳性;(2)hucmsc-mvs的分离和鉴定:经透射电镜观察,显示离心所得产物为一类直径介于40nm-300nm之间大小不一的囊泡状结构,重悬后蛋白质浓度约2.05μg/μl。2.hucmsc-mvs在体外与ra患者pbmc混合培养(1)各实验组均可下调th17细胞及il-17a、il-2、il-1、tnf-α的表达,增加treg细胞表达,差别有统计学意义(p0.05);hucmscs组和中、高浓度mvs组均可上调tgf-β、il-10水平,差异有统计学意义(p0.05);(2)高浓度mvs组在下调th17细胞及il-17a、il-1a、tnf-α的表达和增加Treg细胞及TGF-β、IL-10的表达方面,作用优于低、中浓度MVs组,差异有统计学意义(P0.05);(3)高浓度MVs组和hUC-MSCs组均可下调Th17细胞及IL-17A、IL-1a、TNF-α的表达,增加IL-10的表达,差别无统计学意义(P0.05)。结论:1.本实验室培养的hUCMSCs及分离的hUCMSC-MVs基本符合国际标准;2.hUCMSCs和hUCMSC-MVs均可调节Th17/Treg的失衡,下调Th17细胞及促炎因子IL-17A、IL-1a、IL-2、TNF-α的水平,上调Treg细胞及抗炎因子TGF-β和IL-10的水平;高浓度MVs组的免疫调节作用优于低、中浓度组,且不弱于其母体细胞。
[Abstract]:Background: Rheumatoid Arthritis (RA) is a chronic, systematic, inflammatory autoimmune disease, and its pathological changes are synovial hyperplasia, pannus formation and destruction of bone and cartilage tissue caused by immune dysfunction. Research shows that the imbalance of Treg/Th17 cells plays a crucial role in the development of RA. The important role of [1,2]. Mesenchyma Stem Cells (MSCs) has been proved to be a highly value-added and pluripotent cell [3], which can be used to treat RA by regulating the expression of inflammatory cells and factors. In recent years, the microbubbles (Mesenchymal Stem Cells deri) derived from mesenchymal stem cells (Mesenchymal Stem Cells deri) Ved Microvesicles, MSC-MVs) also has immunomodulatory function [5], at the same time it can avoid the risk of vascular embolism caused by cell therapy, ectopic osteogenesis and so on. Therefore, it has gradually become a new research hotspot. Objective: through the culture of human umbilical cord mesenchymal stem cells (human umbilical cord mesenchymal stem cells, hUC-MSCs) and the separation of human umbilical cord in vitro Microbubbles (human umbilical cord mesenchymal stem cells derived microvesicles, hUCMSC-MVs) from the stem cells were co cultured with the peripheral blood mononuclear cells of hUCMSC-MVs and RA patients. The effect of L-2, TNF- alpha, TGF- beta, IL-10 expression, to explore whether hUCMSC-MVs is effective in the treatment of RA, how its action intensity is related to its concentration, and then explain the possible mechanism of hUCMSC-MVs treatment RA from inflammatory cells and factor pathways. Methods: 1.hUC-MSCs and hUCMSC-MVs separation, identification (1) hUC-MSCs separation, culture and identification: tissue culture and identification: use tissue Block culture method in vitro, culture hUC-MSCs, inverted microscope observation of its morphology, flow cytometry detection of surface antigen CD29, CD44, CD105, CD73, CD90, CD166, CD34, CD45, and under specific conditions, the osteogenesis of hUC-MSCs, identification of lipid differentiation potential; (2) hucmsc-mvs isolation and identification: hUC-MSCs "hunger" culture for 24 hours, use The microbubbles in the supernatant were extracted by differential centrifugation, and the morphology was observed by transmission electron microscopy. The protein content of.2.hucmsc-mvs was measured by the BCA method and the PBMCs co culture of the patients with RA was observed. The effects on the expression of Th17 cells, Treg cells and related inflammatory factors were observed (1): a total of 5 groups were blank control group, huc-m Group SCS (2 x 106), group hucmsc-mvs (30 mu g), group hucmsc-mvs (60 mu g), hucmsc-mvs group (90 mu g). (2) detection index: (1) flow cytometry to determine Th17 cells and Treg cell expression level. 2. Flow cytometry, high throughput and multifactor detection technique, to determine the expression level of IL-17A, IL-1A, IL-2, alpha and anti-inflammatory factors in culture supernatant. Results: statistical analysis. Results: separation of 1.huc-mscs and hucmsc-mvs, identification (1) separation, culture and identification of hUC-MSCs: under inverted microscope, the cells arranged neatly, showing a relatively uniform long spindle shape, similar to fibroblast like appearance. The cell surface CD29, CD44, CD73, CD90, CD105 and CD166 were positive by flow cytometry. CD34, CD45 negative expression.Huc-mscs was induced by osteogenic differentiation and stained positive by calcium cobalt method; after lipid induced differentiation, oil red O staining was positive; (2) separation and identification of hucmsc-mvs: through transmission electron microscopy, the results showed that the product of centrifugation was a kind of vesicular structure with a diameter of different size between 40nm-300nm and the concentration of protein after suspension was about 2. .05 mu g/ mu l.2.hucmsc-mvs in vitro and RA patients with PBMC mixed culture (1) all the experimental groups can reduce the expression of Th17 cells and IL-17A, IL-2, IL-1, tnf- a, increase the expression of Treg cells, the difference is statistically significant (P0.05). The expression of Th17 cells and IL-17A, IL-1A, tnf- alpha and the expression of Treg cells and TGF- beta and IL-10 were better than those in the low concentration MVs group, and the difference was statistically significant (P0.05). (3) the expression of Th17 cells and Th17 cells was down to be down, and there was no significant difference in the expression of Th17 cells and hUC-MSCs. P0.05) conclusion: 1. the hUCMSCs and the isolated hUCMSC-MVs cultured in the laboratory basically conform to the international standards; both 2.hUCMSCs and hUCMSC-MVs can regulate the imbalance of Th17/Treg, down regulate the level of Th17 cells and proinflammatory factors IL-17A, IL-1a, IL-2, TNF- alpha, up regulation of Treg cells and anti-inflammatory cytokines, and the immunoregulation of high concentration group. It is superior to the low, medium concentration group, and not weaker than its maternal cells.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R593.22

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