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

【摘要】： 近年來心肌再生過程和技術(shù)的相關(guān)研究已經(jīng)成為再生醫(yī)學(xué)和心臟病學(xué)研究的熱點,骨髓間充質(zhì)干細(xì)胞(bone marrow mesenchymal stem cells,MSCs)作為細(xì)胞治療最常用候選種子細(xì)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(lǐng)域研究的焦點。但干細(xì)胞移植入缺血性心臟后存活率較低的問題卻成為該技術(shù)應(yīng)用的瓶頸。因此闡明移植后干細(xì)胞凋亡的分子機(jī)制并改善局部微環(huán)境,降低因缺血引起的細(xì)胞死亡及凋亡已成為心臟再生醫(yī)學(xué)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(hù)作用國內(nèi)外尚未見報導(dǎo)。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(hù)作用及作用機(jī)制。 快速自我更新細(xì)胞(rapidly self-renewing cells,RS細(xì)胞)是在探索獲取具有更強(qiáng)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(xì)胞群過程中發(fā)現(xiàn)的一個小細(xì)胞亞群,該類細(xì)胞表達(dá)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(xì)胞治療更具明顯優(yōu)勢。因此,以RS細(xì)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(jìn)一步推進(jìn)RS細(xì)胞生理機(jī)制的研究并完善其臨床應(yīng)用,另一方面為探討雌激素作用機(jī)制提供新的思路。 本實驗分別以大鼠骨髓間充質(zhì)干細(xì)胞rMSCs中的mMSCs和RS細(xì)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(xì)胞活性的影響及雌激素的保護(hù)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(hù)劑,分析其保護(hù)作用能力,并初步探索其作用機(jī)制。此外,著重以RS細(xì)胞為研究對象,獲得高質(zhì)量富含RS細(xì)胞的rMSC樣本,以期加快其臨床應(yīng)用進(jìn)程。 本實驗分別以3-1000個細(xì)胞/cm2接種rMSCs,培養(yǎng)7-14天,期間通過倒置顯微鏡觀察細(xì)胞形態(tài)并進(jìn)行圖像分析,測定生長曲線并進(jìn)行生長動力學(xué)分析,以及通過CFU測定評估細(xì)胞增殖潛能。結(jié)果表明,10個細(xì)胞/cm2為最佳接種密度,所獲得rMSC樣本在培養(yǎng)7-10天時細(xì)胞增殖能力較強(qiáng)。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(xì)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(xì)胞因子。 獲得高質(zhì)量富含RS細(xì)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(xì)胞形態(tài)學(xué)觀察、Hoechest33342熒光染色、Annexin V-PI雙染流式細(xì)胞儀檢測和PI標(biāo)記細(xì)胞周期分析,觀察細(xì)胞形態(tài)變化、核變化、活性/凋亡率變化和對細(xì)胞周期的影響。結(jié)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(xì)胞活性顯著提高(P0.01);并且RS細(xì)胞亞群對SD環(huán)境表現(xiàn)出更強(qiáng)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(hù)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(xì)胞于S期受阻,E2則可以使這一比例降低5.8%。上述結(jié)果表明雌激素可以提高干細(xì)胞自我保護(hù)能力,并且RS細(xì)胞具有更強(qiáng)的自我保護(hù)能力和自我調(diào)節(jié)機(jī)制。此外,推測雌激素在血清剝奪條件下對MSC的保護(hù)作用可能包括通過核受體調(diào)控細(xì)胞周期,以及與細(xì)胞表面受體等非轉(zhuǎn)錄途徑有關(guān)。
[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(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R329

【共引文獻(xiàn)】

相關(guān)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(xì)胞的研究進(jìn)展[J];解剖學(xué)雜志;2009年02期

2 胡詩宇;楊柳;孫海英;;關(guān)于骨髓間充質(zhì)干/祖細(xì)胞群中RS細(xì)胞亞群的研究進(jìn)展[J];生物醫(yī)學(xué)工程學(xué)雜志;2009年04期

相關(guān)博士學(xué)位論文 前5條

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

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

3 徐輝;骨髓干細(xì)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(xué)院;2011年

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

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

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本文編號：2094884