本文選題：骨髓間充質(zhì)干細胞 + RS細胞　； 參考：《上海交通大學》2009年碩士論文

【摘要】： 近年來心肌再生過程和技術的相關研究已經(jīng)成為再生醫(yī)學和心臟病學研究的熱點,骨髓間充質(zhì)干細胞(bone marrow mesenchymal stem cells,MSCs)作為細胞治療最常用候選種子細胞之一,由于具有分化成多種非造血組織的能力而成為該領域研究的焦點。但干細胞移植入缺血性心臟后存活率較低的問題卻成為該技術應用的瓶頸。因此闡明移植后干細胞凋亡的分子機制并改善局部微環(huán)境,降低因缺血引起的細胞死亡及凋亡已成為心臟再生醫(yī)學研究的重要課題。 有研究表明雌激素在缺血性損傷后的心肌重塑過程中發(fā)揮重要作用,但目前體外雌激素對心肌缺血引起的MSC壞死和凋亡是否具有保護作用國內(nèi)外尚未見報導。本研究的目的正在于探討雌激素在缺血環(huán)境中對MSCs的保護作用及作用機制。 快速自我更新細胞(rapidly self-renewing cells,RS細胞)是在探索獲取具有更強自我更新能力及多向分化潛能均質(zhì)骨髓間充質(zhì)干/祖細胞群過程中發(fā)現(xiàn)的一個小細胞亞群,該類細胞表達與成熟MSCs(mature MSCs,mMSCs)不同的蛋白及表面抗原,用于體內(nèi)長期移植或細胞治療更具明顯優(yōu)勢。因此,以RS細胞作為本研究的研究對象建立體外血清剝奪模型模擬心肌缺血環(huán)境,一方面進一步推進RS細胞生理機制的研究并完善其臨床應用,另一方面為探討雌激素作用機制提供新的思路。 本實驗分別以大鼠骨髓間充質(zhì)干細胞rMSCs中的mMSCs和RS細胞亞群為研究對象,探討血清剝奪體外培養(yǎng)模擬心臟缺血環(huán)境對不同MSC亞群細胞活性的影響及雌激素的保護作用。本文的創(chuàng)新點在于第一次使用雌激素作為心臟缺血環(huán)境中MSCs的保護劑,分析其保護作用能力,并初步探索其作用機制。此外,著重以RS細胞為研究對象,獲得高質(zhì)量富含RS細胞的rMSC樣本,以期加快其臨床應用進程。 本實驗分別以3-1000個細胞/cm2接種rMSCs,培養(yǎng)7-14天,期間通過倒置顯微鏡觀察細胞形態(tài)并進行圖像分析,測定生長曲線并進行生長動力學分析,以及通過CFU測定評估細胞增殖潛能。結果表明,10個細胞/cm2為最佳接種密度,所獲得rMSC樣本在培養(yǎng)7-10天時細胞增殖能力較強。并且發(fā)現(xiàn)隨著接種密度的升高,rMSC增殖能力下降的越快。此外,mMSC對RS細胞增殖產(chǎn)生抑制作用,可能同時通過接觸抑制和分泌某些細胞因子。 獲得高質(zhì)量富含RS細胞的rMSC樣本后,本實驗以無血清體外培養(yǎng)rMSCs為模型,經(jīng)細胞形態(tài)學觀察、Hoechest33342熒光染色、Annexin V-PI雙染流式細胞儀檢測和PI標記細胞周期分析,觀察細胞形態(tài)變化、核變化、活性/凋亡率變化和對細胞周期的影響。結果顯示SD 12小時后rMSC皺縮變圓、染色質(zhì)固縮,出現(xiàn)凋亡各階段形態(tài)特征;與對照組相比mMSC及RS活性明顯降低并且凋亡率升高(P均小于0.01),與血清剝奪組相比添加10nM E2后細胞活性顯著提高(P0.01);并且RS細胞亞群對SD環(huán)境表現(xiàn)出更強的耐受性,與mMSC亞群相比存活率提高2.75倍,凋亡率降低近三分之一,并在雌激素的保護作用下繼續(xù)保持良好增殖。同時還發(fā)現(xiàn)SD環(huán)境使細胞于S期受阻,E2則可以使這一比例降低5.8%。上述結果表明雌激素可以提高干細胞自我保護能力,并且RS細胞具有更強的自我保護能力和自我調(diào)節(jié)機制。此外,推測雌激素在血清剝奪條件下對MSC的保護作用可能包括通過核受體調(diào)控細胞周期,以及與細胞表面受體等非轉錄途徑有關。
[Abstract]:Bone marrow mesenchymal stem cells ( MSCs ) , as one of the most commonly used candidate seed cells for cell therapy , has become the focus of the research in the field of regenerative medicine and cardiologist . However , the problem of low survival rate of bone marrow mesenchymal stem cells ( MSCs ) has become the bottleneck in the field .



It has been shown that estrogen plays an important role in the process of myocardial remodeling after ischemic injury , but the effect of estrogen on myocardial ischemia - induced MSC necrosis and apoptosis has not been reported at home and abroad . The purpose of this study is to investigate the protective effect and mechanism of estrogen on MSCs in ischemic environment .



Rapid self - renewal cells ( RS cells ) are a small subset of cells found in mesenchymal stem / progenitor cells with stronger self - renewal ability and multi - directional differentiation potential . These cells express different proteins and surface antigens than mature MSCs ( mature MSCs and mMSCs ) and are used in vivo long - term transplantation or cell therapy . Therefore , using RS cells as the research object of this study establishes an in vitro serum deprivation model to simulate the myocardial ischemia environment . On the one hand , the study of the physiological mechanism of RS cells is further advanced and its clinical application is improved . On the other hand , new ideas are provided to explore the mechanism of estrogen action .



In this experiment , the mMSCs and RS cell subsets of rat bone marrow mesenchymal stem cells ( rMSCs ) were studied . The effects of serum deprivation on the activity of different MSC subgroups and the protective effects of estrogen were discussed . The innovative point of this paper was to use estrogen as the protective agent of MSCs in the ischemic environment of the heart for the first time , to analyze its protective effect and to explore its mechanism of action . In addition , the rMSC samples with high quality RS - rich cells were obtained with RS cells as the research subjects , with a view to accelerating its clinical application process .



In this experiment , rMSCs were seeded with 3 - 1000 cells / cm2 and cultured for 7 - 14 days . The cell morphology was observed by an inverted microscope . The growth curve was measured and the growth kinetics of the cells was evaluated . The results showed that 10 cells / cm2 were the best seeded density , the faster the cell proliferation ability was obtained with the increase of the inoculation density , the faster the rMSC proliferation ability decreased . In addition , the mMSC inhibited the proliferation of RS cells , which could inhibit and secrete some cytokines at the same time .



The results showed that estrogen can improve the self - protection ability of the stem cells and increase the apoptosis rate ( P < 0.01 ) .
【學位授予單位】：上海交通大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R329

【共引文獻】

相關期刊論文 前2條

1 魏顯招;張傳森;溫昱;張志英;;極小胚胎樣干細胞的研究進展[J];解剖學雜志;2009年02期

2 胡詩宇;楊柳;孫海英;;關于骨髓間充質(zhì)干/祖細胞群中RS細胞亞群的研究進展[J];生物醫(yī)學工程學雜志;2009年04期

相關博士學位論文 前5條

1 李震;源自經(jīng)培養(yǎng)間充質(zhì)干細胞的轉化細胞群的研究[D];暨南大學;2010年

2 包新杰;移植人骨髓間充質(zhì)干細胞促進大鼠腦梗死后神經(jīng)再生及功能恢復[D];北京協(xié)和醫(yī)學院;2011年

3 徐輝;骨髓干細胞優(yōu)化移植治療急性心肌梗死的實驗研究[D];北京協(xié)和醫(yī)學院;2011年

4 肖慶;小鼠視網(wǎng)膜多潛能干細胞的分離培養(yǎng)和小鼠胚胎干細胞向視網(wǎng)膜色素上皮細胞定向分化的研究[D];中南大學;2010年

5 徐巍;骨髓間充質(zhì)干細胞對光損傷視網(wǎng)膜的保護作用與機制[D];福建醫(yī)科大學;2013年

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本文編號：2094884