發(fā)布時間：2018-04-15 13:02

  本文選題：骨髓間充質(zhì)干細(xì)胞 + 分化��； 參考：《復(fù)旦大學(xué)》2009年博士論文

【摘要】： 骨髓干細(xì)胞是一類具有高度自我更新和多向分化潛能的細(xì)胞，利用其可分化為多種細(xì)胞的能力，可作為受損心肌組織修復(fù)的供體細(xì)胞。骨髓間充質(zhì)干細(xì)胞(mesenchymal stem cells，MSCs)是骨髓中除造血干細(xì)胞以外的另一類多能干細(xì)胞，可能成為一種理想的修復(fù)損傷心肌的移植用細(xì)胞，其治療缺血性心臟病、擴(kuò)張性心肌病等原因引起的心功能不全的研究尚處于起步階段，，正日益受到關(guān)注。本研究以人MSCs為研究對象，探討其增殖、旁分泌、遷移、凋亡過程中不同藥物的影響，及相關(guān)的細(xì)胞信號傳導(dǎo)途徑。 第一部分人骨髓間充質(zhì)干細(xì)胞的分離培養(yǎng)、鑒定及誘導(dǎo)分化 [目的]分離并培養(yǎng)人MSCs，純化擴(kuò)增，為進(jìn)一步進(jìn)行藥物干預(yù)研究打基礎(chǔ)，并檢測細(xì)胞表型。使用5-氮雜胞苷(5-azacytidine，5-aza)將人MSCs向心肌細(xì)胞方向誘導(dǎo)分化，進(jìn)行細(xì)胞鑒定，以滿足實(shí)驗(yàn)的需要。 [方法]抽取志愿者骨髓液，使用密度梯度離心法分離骨髓單個核細(xì)胞，并借助MSCs黏附于塑料瓶底這一特性進(jìn)行純化。相差顯微鏡觀察形態(tài)變化，流式細(xì)胞儀檢測CD34、CD105、CD106表面抗原表達(dá)，鑒定MSCs。將MSCs傳代擴(kuò)增培養(yǎng)，取第6代MSCs經(jīng)5-aza誘導(dǎo)分化，相差顯微鏡觀察形態(tài)變化，免疫化學(xué)鑒定細(xì)胞誘導(dǎo)前后肌球蛋白重鏈β和心肌肌鈣蛋白T(cardiac troponin T，cTnT)表達(dá)。 [結(jié)果]將密度梯度離心法與貼壁法相結(jié)合，可獲得較多的MSCs，通過傳代后細(xì)胞進(jìn)一步純化，每個25cm~2細(xì)胞培養(yǎng)瓶接種2×10~5個細(xì)胞，完全培養(yǎng)液胎牛血清濃度為10％，80％～90％細(xì)胞融合后按1：3或1：2比例傳代，MSCs能穩(wěn)定地傳代擴(kuò)增而無明顯分化跡象。原代細(xì)胞培養(yǎng)2周左右可首次傳代，以后7d左右傳代一次，傳到第6代未發(fā)現(xiàn)屬性改變，呈現(xiàn)成纖維細(xì)胞樣生長。應(yīng)用5-aza對第6代細(xì)胞進(jìn)行誘導(dǎo)分化2周，發(fā)現(xiàn)細(xì)胞形態(tài)變大，連接緊密，呈條索狀，外表似心肌細(xì)胞，誘導(dǎo)前MSCs肌球蛋白重鏈β和cTnT表達(dá)均陰性，誘導(dǎo)后則均為陽性表達(dá)，陽性細(xì)胞率約為21％。 [結(jié)論]應(yīng)用密度梯度離心法密度梯度離心法與貼壁法相結(jié)合，可建立人MSCs體外穩(wěn)定的純化擴(kuò)增方法。體外5-aza誘導(dǎo)后2周，MSCs從形態(tài)和蛋白表達(dá)水平上向心肌細(xì)胞方向分化，符合實(shí)驗(yàn)要求。 第二部分促紅細(xì)胞生成素、普伐他汀、紅景天苷、抗血小板藥物對人骨髓間充質(zhì)干細(xì)胞增殖、旁分泌和分化的干預(yù)研究 [目的]觀察重組人促紅細(xì)胞生成素(recombinant human EPO，rhEPO)、普伐他汀、紅景天苷、抗血小板藥物干預(yù)對人MSCs增殖、血管內(nèi)皮生長因子(vascularendothelial growth factor，VEGF)分泌、分化的影響。 [方法]體外擴(kuò)增培養(yǎng)人MSCs，繪制生長曲線，使用上述藥物干預(yù)第6代人MSCs，相差顯微鏡觀察藥物作用后細(xì)胞形態(tài)學(xué)變化，四甲基偶氮唑鹽[3-(4，5-dimethylthiazol-2-yl)-2，5-diphenyltetrazolium bromide，MTT]比色法檢測細(xì)胞增殖情況，酶聯(lián)免疫吸附法(enzyme-linked immunosorbent assay，ELISA)法測定細(xì)胞VEGF分泌，流式細(xì)胞儀檢測細(xì)胞抗原CD34、CD105、CD106表達(dá)變化，免疫化學(xué)觀察藥物預(yù)處理后經(jīng)5-aza誘導(dǎo)，人MSCs cTnT抗體染色陽性細(xì)胞率的變化。 [結(jié)果](1)細(xì)胞增殖情況：傳代細(xì)胞潛伏期為48h左右，對數(shù)增殖期約為接種后第4～6d，至接種后第7d進(jìn)入平臺期。經(jīng)藥物作用后的人MSCs形態(tài)和細(xì)胞表面抗原CD34、CD105、CD106表達(dá)無明顯變化。反映MSCs增殖變化的OD570值在一定濃度rhEPO(0．01U／mL～10U／mL)、普伐他汀(0．2μmol／L～10μmol／L)、紅景天苷(0．2mg／L～2mg／L)、氯吡格雷(0．02μmol／L～40μmol／L)和噻氯匹定(0．02μmol／L～40μmol／L)作用后明顯高于對照組(P＜0．05)，而部分濃度的阿司匹林組(60μmol／L～2000μmol／L)則顯著低于對照組(P＜0．05)。(2)細(xì)胞VEGF分泌：人MSCs分泌的VEGF水平在高濃度紅景天苷(5mg／L)、高濃度氯吡格雷(40μmol／L)和噻氯匹定(40μmol／L)作用后明顯低于對照組(P＜0．01)，而低濃度普伐他汀(0．2μmol／L)、阿司匹林組和低濃度氯吡格雷組(0．02μmol／L)VEGF水平與對照組無明顯差異，rhEPO、高濃度普伐他汀(100μmol／L)、低濃度紅景天苷(0．2mg／L)和低濃度噻氯匹定(0．02μmol／L)VEGF水平則顯著高于對照組。(3)細(xì)胞分化情況：細(xì)胞表面抗原CD34、CD105、CD106在6種藥物作用后與對照組相比無明顯差異。促紅細(xì)胞生成素、普伐他汀、紅景天苷、氯吡格雷、噻氯匹定和阿司匹林預(yù)處理后經(jīng)5-aza誘導(dǎo)，人MSCs cTnT單克隆抗體染色陽性細(xì)胞百分率無明顯變化(P值分別為0．597、0．952、0．325、0．141、0．830和0．072)。 [結(jié)論]rhEPO、普伐他汀、紅景天苷、氯吡格雷和噻氯匹定對人MSCs有明顯的促進(jìn)增殖作用，而阿司匹林則抑制其增殖。高濃度紅景天苷、氯吡格雷和噻氯匹定有抑制人MSCs分泌VEGF的作用，rhEPO、高濃度普伐他汀、低濃度紅景天苷和低濃度噻氯匹定可促進(jìn)VEGF分泌。這些藥物對人MSCs的分化影響不明顯。 第三部分：促紅細(xì)胞生成素和普伐他汀對人骨髓間充質(zhì)干細(xì)胞增殖、旁分泌、凋亡、遷移和黏附的影響和作用機(jī)制的實(shí)驗(yàn)研究 [目的]觀察促紅細(xì)胞生成素和普伐他汀干預(yù)對人MSCs增殖、VEGF分泌、凋亡、遷移和黏附的影響及相關(guān)細(xì)胞信號傳導(dǎo)通路。 [方法]體外培養(yǎng)人MSCs，使用上述藥物干預(yù)第6代人MSCs，Western blot方法檢測作用前后磷酸化細(xì)胞外信號調(diào)節(jié)激酶1／2(extracellular signal-regulated kinase1／2，ERK1／2)、p38促分裂原活化蛋白激酶(p38 mitogen activated protein kinase，p38MAPK)及磷脂酰肌醇-3-激酶(phosphatidylinositol-3-kinase，PI3K)／絲氨酸／蘇氨酸激酶(serine／threonine kinase，Akt)蛋白的表達(dá)情況，進(jìn)一步用特異的細(xì)胞信號通路抑制劑或激動劑阻斷或激活ERK1／2、p38MAPK及PI3K／AKT途徑，通過MTT法測定對人MSCs的增殖影響，ELISA法測定細(xì)胞VEGF的分泌，用流式細(xì)胞儀進(jìn)行rhEPO和普伐他汀對MSCs的抗凋亡研究，Transwell小室進(jìn)行細(xì)胞遷移實(shí)驗(yàn)，并進(jìn)行細(xì)胞黏附性測定。 [結(jié)果]rhEPO和普伐他汀可使人MSCs的PI3K／Akt通路磷酸化水平升高，抑制p38MAPK通路磷酸化水平，對人MSCs的ERK通路和總Akt、總p38MAPK水平無明顯影響。經(jīng)p38MAPK通路特異激動劑anisomycin預(yù)處理后，rhEPO組促增殖作用較前減弱(P＜0．05)，而普伐他汀組作用消失，PI3K／AKT通路特異抑制劑Ly294002預(yù)處理影響不明顯。Ly294002或anisomycin預(yù)處理均可使rhEPO促進(jìn)人MSCs分泌VEGF的作用減弱(P＜0．01)，Ly294002預(yù)處理可使普伐他汀的這種作用減弱(P＜0．01)，但anisomycin作用不明顯。rhEPO和普伐他汀能降低H_2O_2誘導(dǎo)的人MSCs凋亡(P＜0．01)，Ly294002預(yù)處理后這種作用消失，anisomycin預(yù)處理對這種作用影響不明顯。經(jīng)rhEPO或普伐他汀作用后遷移細(xì)胞顯著增多(P＜0．01)，Ly294002預(yù)處理后這種作用消失，anisomycin預(yù)處理對這種作用影響不明顯。經(jīng)rhEPO或普伐他汀作用后貼壁細(xì)胞顯著增多(P＜0．01)，但Ly294002或anisomycin預(yù)處理對這種作用影響均不明顯。 [結(jié)論]rhEPO及普伐他汀可促進(jìn)人MSCs的增殖、分泌VEGF、遷移和黏附能力，并具有抗凋亡作用。這兩種藥物的促進(jìn)增殖作用與其抑制p38MAPK通路有關(guān)，p38MAPK通路受抑制也與rhEPO促進(jìn)VEGF分泌相關(guān)，而PI3K／Akt通路的激活參與了它們的刺激VEGF分泌、增強(qiáng)遷移能力以及抗凋亡作用的調(diào)控。這兩種藥物促進(jìn)黏附能力與PI3K／Akt和p38MAPK通路無關(guān)。
[Abstract]:Bone marrow stem cells are a kind of highly self-renewal and pluripotent cells, which can differentiate into a variety of cells, can be used as donor cells to repair damaged myocardial tissue. Bone marrow mesenchymal stem cells (mesenchymal stem cells, MSCs) bone marrow mesenchymal stem cells outside of another type of pluripotent stem cells may be an ideal repair of myocardial injury by cell transplantation in the treatment of ischemic heart disease, heart function, study the cause of dilated cardiomyopathy insufficiency is still in its infancy, is getting more and more attention. The research on MSCs as the research object, to explore the proliferation, paracrine, migration. Effect of different drugs in the process of apoptosis, and cell related signal pathways.
Part one, isolation, identification and differentiation of human bone marrow mesenchymal stem cells
[Objective] to isolate and culture human MSCs, purify and amplify it, lay the foundation for further drug intervention research, and detect cell phenotypes. 5- 5-azacytidine (5-aza) is used to induce the differentiation of human MSCs into cardiomyocytes, and cell identification is performed to meet the needs of experiment.
[Methods] from volunteer bone marrow, bone marrow mononuclear cells were isolated by density gradient centrifugation, and with the help of MSCs adhesion to the plastic bottom this characteristic were purified. Morphologic changes were observed by phase contrast microscopy, CD34 assay, flow cytometry and CD105, expression of CD106 surface antigen, MSCs. MSCs identification of cultured passaged sixth generations MSCs by 5-aza induced differentiation, morphologic changes were observed by phase contrast microscope, immunohistochemical identification of cells before and after induction of beta myosin heavy chain and cardiac troponin T (cardiac troponin T, cTnT) expression.
[results] the combination of density gradient centrifugation and adherent method can obtain more MSCs, subcultured cells was further purified by 25cm~2, each cell culture bottle with 2 * 10~5 cells, completely cultured fetal bovine serum concentration was 10%, from 80% to 90% after cell fusion by 1:3 or 1:2 cases than the passage MSCs can be stably passaged and amplified without obvious signs of differentiation. The primary cell culture 2 weeks after the first passage, a passage about 7d, reached the sixth generation did not find the property change, showing fibroblastic growth. Application of 5-aza to the sixth generation cells were differentiated for 2 weeks, found that the cells became larger, connection close a funicular look like myocardial cells before induction of MSCs myosin heavy chain and beta cTnT expression were negative after induction were positive, positive rate is about 21%.
[Conclusion] the combination of density gradient centrifugation and density gradient centrifugation combined with adherence can establish a stable amplification method for human MSCs in vitro. After 2 weeks of 5-aza induction, MSCs will differentiate into cardiomyocytes from morphology and protein expression, which is in line with the experimental requirements.
Second parts of erythropoietin, pravastatin, salidroside, antiplatelet drugs on the proliferation, paracrine and differentiation of human bone marrow mesenchymal stem cells
[Objective] to observe the effects of recombinant human EPO (rhEPO), pravastatin and salidroside on the proliferation of MSCs, the secretion and differentiation of vascularendothelial growth factor (VEGF) in human MSCs.
[Methods] human MSCs cultured in vitro, draw the growth curve, the use of drug intervention and sixth generations of MSCs, phase contrast microscope and morphological changes were observed after treatment with four methyl thiazolyl tetrazolium [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide, MTT] colorimetric assay for cell proliferation, enzyme linked immunosorbent assay (enzyme-linked immunosorbent assay, ELISA) method for the determination of cell secretion of VEGF cell antigen detection, CD34, flow cytometry, CD105, expression of CD106, immunohistochemical observation of drug pretreatment induced by 5-aza MSCs cTnT, changes in antibody staining positive cell rate.
[緇撴灉](1)緇嗚優(yōu)澧炴畺鎯呭喌錛氫紶浠ｇ粏鑳?yōu)娼滀紡鏈熶?

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