發(fā)布時(shí)間：2018-06-17 17:24

  本文選題：類風(fēng)濕關(guān)節(jié)炎 + 間充質(zhì)干細(xì)胞微泡��； 參考：《山西醫(yī)科大學(xué)》2017年碩士論文

【摘要】：背景:類風(fēng)濕關(guān)節(jié)炎(Rheumatoid Arthritis,RA)是一種慢性、系統(tǒng)性、炎癥性自身免疫性疾病,其病理改變是機(jī)體免疫功能紊亂導(dǎo)致的滑膜增生、血管翳形成、骨及軟骨組織的破壞。有研究的表明,Treg/Th17細(xì)胞的平衡失調(diào)對(duì)RA的發(fā)生、發(fā)展起著至關(guān)重要的作用[1,2]。間充質(zhì)干細(xì)胞(Mesenchyma Stem Cells,MSCs)已被證實(shí)是一種具有高度增值能力和多向分化潛能的細(xì)胞[3],可通過(guò)調(diào)節(jié)炎癥細(xì)胞及因子的表達(dá),達(dá)到治療RA的目的。近幾年研究發(fā)現(xiàn),間充質(zhì)干細(xì)胞來(lái)源的微泡(Mesenchymal Stem Cells derived Microvesicles,MSC-MVs)同樣具有免疫調(diào)節(jié)作用[5],同時(shí)可避免細(xì)胞治療引起的血管栓塞、異位成骨等風(fēng)險(xiǎn),因此逐漸成為新型的研究熱點(diǎn)。目的:通過(guò)體外培養(yǎng)人臍帶間充質(zhì)干細(xì)胞(human umbilical cord mesenchymal stem cells,hUC-MSCs)及分離人臍帶間充質(zhì)干細(xì)胞來(lái)源的微泡(human umbilical cord mesenchymal stem cells derived microvesicles,hUCMSC-MVs),將hUCMSC-MVs與RA患者外周血單個(gè)核細(xì)胞(Peripheral Blood Mononuclear Cells,PBMCs)體外共培養(yǎng),觀察hUCMSC-MVs對(duì)RA患者PBMC中Th17細(xì)胞、Treg細(xì)胞及炎性因子IL-17A、IL-1a、IL-2、TNF-α、TGF-β、IL-10表達(dá)的影響,探討hUCMSC-MVs治療RA是否有效,作用強(qiáng)度如何,是否與其濃度相關(guān),進(jìn)而從炎癥細(xì)胞及因子途徑闡述hUCMSC-MVs治療RA可能的作用機(jī)制。方法:1.hUC-MSCs和hUCMSC-MVs的分離、鑒定(1)huc-mscs的分離、培養(yǎng)和鑒定:采用組織塊培養(yǎng)方法體外分離、培養(yǎng)huc-mscs,倒置顯微鏡下觀察其形態(tài),流式細(xì)胞術(shù)檢測(cè)表面抗原cd29、cd44、cd105、cd73、cd90、cd166、cd34、cd45,并在特定條件下對(duì)huc-mscs的成骨、成脂分化潛能進(jìn)行鑒定;(2)hucmsc-mvs的分離和鑒定:huc-mscs“饑餓”培養(yǎng)24小時(shí),使用差速離心提取上清液中的微泡,使用透射電鏡觀察其形態(tài),并采用bca方法在酶標(biāo)儀上測(cè)定其蛋白質(zhì)含量。2.hucmsc-mvs與ra患者pbmcs共培養(yǎng),觀察其對(duì)th17細(xì)胞、treg細(xì)胞及相關(guān)炎性因子表達(dá)水平的影響(1)實(shí)驗(yàn)分組:共5組,分別是空白對(duì)照組、huc-mscs組(2×106個(gè))、hucmsc-mvs組(30μg)、hucmsc-mvs組(60μg)、hucmsc-mvs組(90μg)。(2)檢測(cè)指標(biāo):⑴流式細(xì)胞術(shù)測(cè)定th17細(xì)胞、treg細(xì)胞表達(dá)水平。⑵流式高通量多因子檢測(cè)技術(shù)測(cè)定培養(yǎng)上清中促炎因子il-17a、il-1a、il-2、tnf-α和抗炎因子tgf-β、il-10的表達(dá)水平,收集數(shù)據(jù)做統(tǒng)計(jì)分析。結(jié)果:1.huc-mscs和hucmsc-mvs的分離、鑒定(1)huc-mscs的分離、培養(yǎng)和鑒定:倒置顯微鏡下觀察細(xì)胞排列較整齊,呈較為均一的長(zhǎng)梭形,類似成纖維細(xì)胞樣外觀。經(jīng)流式細(xì)胞術(shù)檢測(cè),細(xì)胞表面cd29、cd44、cd73、cd90、cd105和cd166呈陽(yáng)性表達(dá),cd34、cd45成陰性表達(dá)。huc-mscs經(jīng)成骨誘導(dǎo)分化,鈣鈷法染色成陽(yáng)性;經(jīng)成脂誘導(dǎo)分化,油紅o染色成陽(yáng)性;(2)hucmsc-mvs的分離和鑒定:經(jīng)透射電鏡觀察,顯示離心所得產(chǎn)物為一類直徑介于40nm-300nm之間大小不一的囊泡狀結(jié)構(gòu),重懸后蛋白質(zhì)濃度約2.05μg/μl。2.hucmsc-mvs在體外與ra患者pbmc混合培養(yǎng)(1)各實(shí)驗(yàn)組均可下調(diào)th17細(xì)胞及il-17a、il-2、il-1、tnf-α的表達(dá),增加treg細(xì)胞表達(dá),差別有統(tǒng)計(jì)學(xué)意義(p0.05);hucmscs組和中、高濃度mvs組均可上調(diào)tgf-β、il-10水平,差異有統(tǒng)計(jì)學(xué)意義(p0.05);(2)高濃度mvs組在下調(diào)th17細(xì)胞及il-17a、il-1a、tnf-α的表達(dá)和增加Treg細(xì)胞及TGF-β、IL-10的表達(dá)方面,作用優(yōu)于低、中濃度MVs組,差異有統(tǒng)計(jì)學(xué)意義(P0.05);(3)高濃度MVs組和hUC-MSCs組均可下調(diào)Th17細(xì)胞及IL-17A、IL-1a、TNF-α的表達(dá),增加IL-10的表達(dá),差別無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論:1.本實(shí)驗(yàn)室培養(yǎng)的hUCMSCs及分離的hUCMSC-MVs基本符合國(guó)際標(biāo)準(zhǔn);2.hUCMSCs和hUCMSC-MVs均可調(diào)節(jié)Th17/Treg的失衡,下調(diào)Th17細(xì)胞及促炎因子IL-17A、IL-1a、IL-2、TNF-α的水平,上調(diào)Treg細(xì)胞及抗炎因子TGF-β和IL-10的水平;高濃度MVs組的免疫調(diào)節(jié)作用優(yōu)于低、中濃度組,且不弱于其母體細(xì)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.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R593.22

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