本文關(guān)鍵詞： 脂肪干細(xì)胞 細(xì)胞治療 輔助移植 Edu 干細(xì)胞標(biāo)記 細(xì)胞示蹤　出處：《北京協(xié)和醫(yī)學(xué)院》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：研究背景： 人脂肪干細(xì)胞(ADSC,Adipose-derived stem cells)是從脂肪組織中分離出來的,并能顯示出持續(xù)的細(xì)胞增殖能力及向不組織細(xì)胞定向分化的能力的細(xì)胞,具有組織量多,來源廣泛的優(yōu)點(diǎn)。近年來,被廣泛地應(yīng)用于基礎(chǔ)研究及臨床細(xì)胞治療中,且具有巨大的發(fā)展?jié)摿�。自體脂肪移植在整形外科中廣泛應(yīng)用,但成活率低,需重復(fù)治療等缺點(diǎn)是整形外科醫(yī)生面臨的重大問題。為了解決這一問題,需要對(duì)脂肪成活機(jī)制準(zhǔn)確把握。在脂肪成活過程中,ADSC的作用尤為明顯。一些理論推斷脂肪干細(xì)胞的作用通過脂肪干細(xì)胞的增殖、分化成新的脂肪組織并提供適應(yīng)的環(huán)境來促進(jìn)脂肪的成活,但未有定論,且需要更多、更準(zhǔn)確的實(shí)驗(yàn)來回答這一問題。我們的實(shí)驗(yàn)選用有種新的示蹤劑,通過細(xì)胞示蹤來探究脂肪干細(xì)胞在脂肪移植中的作用機(jī)制。Edu是一種核酸類似物,在細(xì)胞分裂增殖的過程中參與到核染色體中達(dá)到標(biāo)記目的。 目的： 觀察體外培養(yǎng)時(shí)不同培養(yǎng)階段的人脂肪干細(xì)胞的細(xì)胞形態(tài)和生物學(xué)特點(diǎn)。通過實(shí)驗(yàn)初步確立最佳的標(biāo)記濃度及標(biāo)記時(shí)間組合。進(jìn)一步交通過與未標(biāo)記的干細(xì)胞的進(jìn)行增殖及分化影響的比較以選出最適標(biāo)記濃度及時(shí)間。 方法： 1、人ADSC的體外培養(yǎng)分離、培養(yǎng)和鑒定。經(jīng)知隋同意后,取吸脂手術(shù)來源的脂肪顆粒,使用酶消化法獲得SVF細(xì)胞,原代培養(yǎng)在7-10天時(shí)達(dá)80%-90%的細(xì)胞融合,干細(xì)胞進(jìn)一步傳代培養(yǎng)進(jìn)行純化和擴(kuò)增。倒置顯微鏡下觀察細(xì)胞形態(tài)變化。細(xì)胞傳至第3代后,分別用于鑒定細(xì)胞及用于細(xì)胞標(biāo)記的研究。 2、Edu在不同濃度及時(shí)間標(biāo)記的組合下進(jìn)行研究。分別采5uM,10uM,20uM,50uM的濃度及12小時(shí),24小時(shí)進(jìn)行標(biāo)記,Edu標(biāo)記后的細(xì)胞去除含Edu的培養(yǎng)基,制成單細(xì)胞懸液并固定細(xì)胞,以Alexa-555染色后行流式細(xì)胞分析儀檢測(cè)標(biāo)記率,從而挑選出各時(shí)間點(diǎn)中較適宜的標(biāo)記組合。 3、測(cè)定Edu在較適宜的標(biāo)記率情況下對(duì)細(xì)胞生長及分化的影響,以未標(biāo)記細(xì)胞作為空白對(duì)照。Edu對(duì)干細(xì)胞細(xì)胞活性及增殖的影響以臺(tái)盼藍(lán)排斥反應(yīng)及MTT實(shí)驗(yàn)證實(shí)。 4、測(cè)定Edu標(biāo)記后影響細(xì)胞分化的能力。即去除Edu后,脂肪干細(xì)胞行定向成脂及成骨誘導(dǎo),誘導(dǎo)結(jié)束后行油紅O染色及茜素紅染色鑒定分化結(jié)果。數(shù)據(jù)以SPSS13.0進(jìn)行分析。 結(jié)果： 原代細(xì)胞貼壁后呈一定方向的成纖維細(xì)胞樣生長。在第5到第7天時(shí)細(xì)胞克隆逐漸變大并相互融合并形成單層。7-10天時(shí)將細(xì)胞消化下來并進(jìn)行傳代。傳代后細(xì)胞生長迅速,2-3天即可長滿培養(yǎng)皿的底部并進(jìn)一步傳代。3代細(xì)胞行流式細(xì)胞儀分析細(xì)胞表型時(shí)表達(dá)細(xì)胞表面標(biāo)志如CD90,CD105,CD44,而且不表達(dá)或弱表達(dá)細(xì)胞表面標(biāo)志CD34,CD45。 Edu的標(biāo)記是在細(xì)胞分裂增殖時(shí),新合成的細(xì)胞才能被標(biāo)記上。流式結(jié)果表明,分別在10uM,12h和5uM,24h標(biāo)記時(shí),細(xì)胞標(biāo)記率可達(dá)約90%。更高的濃度并不能明顯提高細(xì)胞的標(biāo)記率,且會(huì)對(duì)細(xì)胞活性有影響。因此,選定此兩組作為實(shí)驗(yàn)組,未標(biāo)記細(xì)胞作為對(duì)照組,分析標(biāo)記后對(duì)細(xì)胞增殖分化的影響。 標(biāo)記后的細(xì)胞即可被消化,制成單細(xì)胞懸液。臺(tái)盼藍(lán)排斥實(shí)驗(yàn)數(shù)據(jù)顯示細(xì)胞死亡比例各組間無統(tǒng)計(jì)學(xué)差異。MTT實(shí)驗(yàn)分析,三組間10uM,12h與對(duì)照組曲線較相近,但略低,5uM,24h與對(duì)照組曲線較遠(yuǎn),證實(shí)標(biāo)記物對(duì)細(xì)施增殖有一定的影響,以10uM,12h組標(biāo)記者影響較小。 標(biāo)記后的細(xì)胞去除Edu后定向分化,成脂誘導(dǎo)2周,成骨誘導(dǎo)3周。分別以油紅O染色及茜素紅染色,隨機(jī)每高倍鏡視野下計(jì)數(shù)成脂細(xì)胞數(shù)及鈣結(jié)節(jié)數(shù),數(shù)據(jù)分析后發(fā)現(xiàn)二者無統(tǒng)計(jì)學(xué)意義。 本實(shí)驗(yàn)在體外培養(yǎng)脂肪干細(xì)胞時(shí),確認(rèn)P3代以后的細(xì)胞較為純化化并可以用來實(shí)驗(yàn)分析。應(yīng)用Edu標(biāo)記脂肪干細(xì)胞并首次應(yīng)用流式方法測(cè)定其精確的標(biāo)記率,通過對(duì)其生長及定向分化能力的測(cè)定,得出最適濃度組合10uM,12h,對(duì)進(jìn)一步研究脂肪干細(xì)胞在輔助脂肪移植中的作用具有指導(dǎo)意義。
[Abstract]:Research background:
Human adipose derived stem cells (ADSC, Adipose-derived stem cells) was extracted from adipose tissue, and can demonstrate the ability of cell proliferation and sustained to tissue differentiation of cells with tissue volume, wide source of advantages. In recent years, is widely used in basic research and clinical cell therapy, and has great potential for development. Autologous fat transplantation is widely used in plastic surgery, but the survival rate is low, the need to repeat the treatment of defects is a major problem facing the plastic surgeon. In order to solve this problem, need to accurately grasp the survival mechanism of fat in fat. The survival process, the ADSC effect is particularly obvious. Some theories postulate that adipose derived stem cells by adipose derived stem cel proliferation, differentiation of adipose tissue and provide a new adaptation of the environment to promote the survival of fat, but not conclusive, and more Much more accurate experiments to answer this question. Our experiment with a new tracer, tracer through the cell to explore the mechanism of.Edu adipose derived stem cells in fat transplantation is a kind of nucleic acid analogues, in the process of cell proliferation involved in nuclear chromosome markers to reach.
Objective:
In vitro culture of different cell morphology and biological characteristics of stem cell stage of human adipose. By preliminary experiments to determine the optimal marker concentration and marking time combination. Further traffic with unlabeled stem cells to influence the proliferation and differentiation to choose the most suitable markers of concentration and time.
Method錛，

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