發(fā)布時間：2018-05-25 02:26

  本文選題：缺氧 + 去鐵胺　； 參考：《暨南大學(xué)》2011年碩士論文

【摘要】：目的：間充質(zhì)干細胞(Mesenchymal stem cells, MSCs)在治療缺血缺氧性疾病時,其治療效能的發(fā)揮與損傷組織中的缺氧環(huán)境密切相關(guān)。本文旨在探索缺氧對人臍帶MSCs的形態(tài)、超微結(jié)構(gòu)、增殖及蛋白差異表達的影響,篩選MSCs治療缺血缺氧性疾病的新靶點,進而嘗試探索MSCs的進一步臨床應(yīng)用。 方法：(1)膠原酶消化法聯(lián)合貼壁法從人臍帶組織中分離提取MSCs,油紅O染色鑒定細胞成脂分化能力,Von Kossa法鑒定細胞成骨分化能力。(2)流式細胞儀測正常人臍帶MSCs的免疫表型及細胞周期比例。(3)缺氧模擬劑去鐵胺(Deferoxamine, DFO)及氯化鈷(Cobalt chloride, CoCl2)分別處理人臍帶MSCs,原子力顯微鏡(AFM)及透射電鏡(TEM)觀察DFO及CoCl2對人臍帶MSCs形態(tài)及超微結(jié)構(gòu)變化。(4)MTT法測DFO及CoCl2對人臍帶MSCs增殖的影響。(5)流式細胞儀測DFO及CoCl2對人臍帶MSCs細胞周期的影響。(6)雙向凝膠電泳技術(shù)(2-DE)分離CoCl2作用前后的人臍帶MSCs總蛋白,ImageMaster 2D Platinum軟件分析蛋白質(zhì)差異表達點,基質(zhì)輔助激光解析串聯(lián)飛行時間質(zhì)譜對差異表達的蛋白進行鑒定及功能分類。 結(jié)果：(1)人臍帶MSCs的G0/G1期細胞占89.4%,高表達CD29, CD44, CD 105,低表達或不表達CD106, CD40, CD34, CD45, HLA-DR。(2)人臍帶MSCs向脂肪細胞誘導(dǎo)分化,油紅O染色后可見脂滴呈紅色。人臍帶MSCs向成骨細胞誘導(dǎo)分化,Von Kossa染色可見黑色礦化結(jié)節(jié)沉積。(3)DFO及CoCl2處理人臍帶MSCs后,細胞變長,毗鄰的細胞間網(wǎng)狀結(jié)構(gòu)消失,代之以間隙。細胞內(nèi)出現(xiàn)大量空泡狀結(jié)構(gòu),粗面內(nèi)質(zhì)網(wǎng)擴張成池,線粒體嵴擴張。(4)DFO及CoCl2明顯抑制人臍帶MSCs增殖,隨DFO及CoCl2濃度增加,增殖抑制率遞增,120μmol/lDFO組,10μmol/lCoCl2組及100μmol/lCoCl2組與對照組相比差異有統(tǒng)計學(xué)意義(P0.05)。(5)DFO及CoCl2使人臍帶MSCs的G0/G1期細胞比例增加,G2/M/S期細胞比例減少。(6)建立了CoCl2作用人臍帶MSCs的蛋白差異表達譜,鑒定出26個差異表達蛋白,其中11個表達上調(diào),15個表達下調(diào)。 結(jié)論：(1)缺氧模擬劑DFO及CoCl2使人臍帶MSCs形態(tài)及超微結(jié)構(gòu)發(fā)生改變,并通過影響細胞周期而抑制MSCs增殖。(2)CoCl2對人臍帶MSCs蛋白差異表達的影響涉及多蛋白通路,包括糖代謝、核酸代謝、脂類代謝、蛋白質(zhì)代謝和修飾；輔酶與輔基代謝；細胞周期；免疫和防御；細胞結(jié)構(gòu)和運動；信號轉(zhuǎn)導(dǎo)；蛋白靶向和定位；胞內(nèi)蛋白運輸；神經(jīng)細胞的活動；肌肉收縮等多種生物學(xué)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.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R329

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