缺氧对脐带间充质干细胞增殖及差异蛋白表达谱的影响

发布时间：2018-05-25 02:26

  本文选题：缺氧 + 去铁胺　； 参考：《暨南大学》2011年硕士论文

【摘要】：目的：间充质干细胞(Mesenchymal stem cells, MSCs)在治疗缺血缺氧性疾病时,其治疗效能的发挥与损伤组织中的缺氧环境密切相关。本文旨在探索缺氧对人脐带MSCs的形态、超微结构、增殖及蛋白差异表达的影响,筛选MSCs治疗缺血缺氧性疾病的新靶点,进而尝试探索MSCs的进一步临床应用。 方法：(1)胶原酶消化法联合贴壁法从人脐带组织中分离提取MSCs,油红O染色鉴定细胞成脂分化能力,Von Kossa法鉴定细胞成骨分化能力。(2)流式细胞仪测正常人脐带MSCs的免疫表型及细胞周期比例。(3)缺氧模拟剂去铁胺(Deferoxamine, DFO)及氯化钴(Cobalt chloride, CoCl2)分别处理人脐带MSCs,原子力显微镜(AFM)及透射电镜(TEM)观察DFO及CoCl2对人脐带MSCs形态及超微结构变化。(4)MTT法测DFO及CoCl2对人脐带MSCs增殖的影响。(5)流式细胞仪测DFO及CoCl2对人脐带MSCs细胞周期的影响。(6)双向凝胶电泳技术(2-DE)分离CoCl2作用前后的人脐带MSCs总蛋白,ImageMaster 2D Platinum软件分析蛋白质差异表达点,基质辅助激光解析串联飞行时间质谱对差异表达的蛋白进行鉴定及功能分类。 结果：(1)人脐带MSCs的G0/G1期细胞占89.4%,高表达CD29, CD44, CD 105,低表达或不表达CD106, CD40, CD34, CD45, HLA-DR。(2)人脐带MSCs向脂肪细胞诱导分化,油红O染色后可见脂滴呈红色。人脐带MSCs向成骨细胞诱导分化,Von Kossa染色可见黑色矿化结节沉积。(3)DFO及CoCl2处理人脐带MSCs后,细胞变长,毗邻的细胞间网状结构消失,代之以间隙。细胞内出现大量空泡状结构,粗面内质网扩张成池,线粒体嵴扩张。(4)DFO及CoCl2明显抑制人脐带MSCs增殖,随DFO及CoCl2浓度增加,增殖抑制率递增,120μmol/lDFO组,10μmol/lCoCl2组及100μmol/lCoCl2组与对照组相比差异有统计学意义(P0.05)。(5)DFO及CoCl2使人脐带MSCs的G0/G1期细胞比例增加,G2/M/S期细胞比例减少。(6)建立了CoCl2作用人脐带MSCs的蛋白差异表达谱,鉴定出26个差异表达蛋白,其中11个表达上调,15个表达下调。 结论：(1)缺氧模拟剂DFO及CoCl2使人脐带MSCs形态及超微结构发生改变,并通过影响细胞周期而抑制MSCs增殖。(2)CoCl2对人脐带MSCs蛋白差异表达的影响涉及多蛋白通路,包括糖代谢、核酸代谢、脂类代谢、蛋白质代谢和修饰；辅酶与辅基代谢；细胞周期；免疫和防御；细胞结构和运动；信号转导；蛋白靶向和定位；胞内蛋白运输；神经细胞的活动；肌肉收缩等多种生物学功能。
[Abstract]:Aim: the efficacy of mesenchymal stem cells, MSCs) in the treatment of ischemic hypoxic diseases is closely related to the hypoxic environment in injured tissues. The purpose of this study was to explore the effects of hypoxia on the morphology, ultrastructure, proliferation and differential expression of protein in human umbilical cord MSCs, to screen new targets for the treatment of ischemic and hypoxic diseases with MSCs, and to explore the further clinical application of MSCs. Methods MSCs were isolated and extracted from human umbilical cord tissues by collagenase digestion and adherent method. The ability of adipogenic differentiation was evaluated by oil red O staining and the osteogenic differentiation ability was evaluated by Von Kossa) flow cytometry (FCM) was used to detect the immunity of normal human umbilical cord MSCs. Morphologic and ultrastructural changes of human umbilical cord MSCs treated with deferoxamine (DFOO) and Cobalt chloride (CoCl2) were observed by DFO and CoCl2 under transmission electron microscope (TEM) and atomic force microscope (AFM) respectively. Effects of DFO and CoCl2 on the Proliferation of Human umbilical Cord MSCs. (5) flow cytometry was used to detect the effect of DFO and CoCl2 on the cell cycle of human umbilical MSCs. Two-dimensional gel electrophoresis (2-DEE) was used to isolate the differential expression points of human umbilical cord MSCs total protein imageMaster 2D Platinum before and after CoCl2 treatment. Matrix assisted laser desorption tandem time of flight mass spectrometry was used to identify and classify differentially expressed proteins. Results the percentage of G0/G1 phase cells in human umbilical cord MSCs was 89.4.The high expression of CD29, CD44, CD105, low expression or non-expression of CD106, CD40, CD34, CD45, HLA-DR. 2) induced differentiation of human umbilical cord MSCs into adipocytes, and lipid droplets were red after oil red O staining. The differentiation of human umbilical cord MSCs into osteoblast induced by Von Kossa staining showed that the cells became longer and the adjacent intercellular reticular structure disappeared and the space was replaced by the black mineralized nodular deposition. After treated with CoCl2, the cells became longer and the adjacent intercellular reticular structure disappeared. A large number of vacuolated structures appeared in the cells, the rough endoplasmic reticulum expanded into a pool, and the mitochondrial cristal dilatation. DFO and CoCl2 significantly inhibited the proliferation of MSCs in human umbilical cord, which increased with the concentration of DFO and CoCl2. There were significant differences in proliferation inhibition rate between 10 渭 mol/lCoCl2 group and 100 渭 mol/lCoCl2 group compared with the control group. Compared with the control group, the increase of G0/G1 phase cell proportion of MSCs in human umbilical cord and the increase of G0/G1 phase cell proportion of MSCs in human umbilical cord by CoCl2. The protein differential expression profile of MSCs in human umbilical cord induced by CoCl2 was established. 26 differentially expressed proteins were identified, of which 11 were up-regulated and 15 down-regulated. Conclusion the anoxic analogue DFO and CoCl2 can change the morphology and ultrastructure of MSCs in human umbilical cord, and inhibit the proliferation of MSCs by affecting the cell cycle. The effect of CoCl2 on the differential expression of MSCs protein in human umbilical cord involves many protein pathways, including glucose metabolism and nucleic acid metabolism. Keywords lipid metabolism, protein metabolism and modification; coenzyme and coenzyme metabolism; cell cycle; immunity and defense; cell structure and movement; signal transduction; protein targeting and localization; intracellular protein transport; neuronal activity; Muscle contraction and other biological functions.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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