【摘要】：目的：分離提取骨髓間充質(zhì)干細(xì)胞(Bone marrow-derived mesenchymal stem cells, BMSCs)及脂肪間充質(zhì)干細(xì)胞(Adipose-derived mesenchymal stem cells, AMSCs)比較二者生物學(xué)特性及體外增殖能力,并探討三種不同成神經(jīng)誘導(dǎo)方法對(duì)AMSCs向神經(jīng)樣細(xì)胞分化過(guò)程中的影響,提高AMSCs向神經(jīng)樣細(xì)胞分化誘導(dǎo)效率,從而為研究AMSCs向神經(jīng)樣細(xì)胞分化機(jī)制奠定基礎(chǔ)。深入探討成球誘導(dǎo)方法中細(xì)胞內(nèi)鈣離子濃度變化,有助于闡明AMSCs的成神經(jīng)誘導(dǎo)分化機(jī)理,為AMSCs在臨床再生醫(yī)學(xué)上的應(yīng)用提供理論依據(jù)。 方法：(1)采用密度梯度離心法分離BMSCs及酶消化法體外分離AMSCs。檢測(cè)MSCs的表面抗原以及將MSCs進(jìn)行體外成骨誘導(dǎo),成脂誘導(dǎo)對(duì)MSCs的干細(xì)胞特性進(jìn)行鑒定。(2)三種誘導(dǎo)方案誘導(dǎo)AMSCs向神經(jīng)樣細(xì)胞分化,包括化學(xué)誘導(dǎo)方法、鼠腦條件培養(yǎng)基誘導(dǎo)方法、成神經(jīng)球分化誘導(dǎo)方法。觀察誘導(dǎo)中的形態(tài)變化,免疫熒光檢測(cè)神經(jīng)細(xì)胞特異性標(biāo)志物β-Ⅲ-Tubulin, NSE和Nissl的表達(dá)。確定最高效的誘導(dǎo)方法。(3)采取成神經(jīng)球誘導(dǎo)方法對(duì)AMSCs進(jìn)行誘導(dǎo),利用Fluo3/AM對(duì)AMSCs及誘導(dǎo)過(guò)程中的類神經(jīng)細(xì)胞進(jìn)行染色,通過(guò)流式細(xì)胞儀對(duì)AMSCs群體細(xì)胞的鈣離子濃度檢測(cè),并通過(guò)共聚焦顯微鏡確定單個(gè)細(xì)胞中鈣離子濃度的變化,研究細(xì)胞內(nèi)游離鈣離子濃度在AMSCs向神經(jīng)樣細(xì)胞分化過(guò)程中的變化。 結(jié)果：(1)獲得的BMSCs及AMSCs可穩(wěn)定傳至20代以上。表型鑒定結(jié)果為CD13、 CD90陽(yáng)性,CD34、CD45陰性,AMSCs的CD106陰性,BMSCs CD106表達(dá)弱陽(yáng)性。成骨細(xì)胞分化28d時(shí),Von kossa陽(yáng)性,向脂肪細(xì)胞分化14d時(shí),油紅O染色陽(yáng)性。(2)用化學(xué)因子誘導(dǎo)AMSCs向神經(jīng)樣細(xì)胞分化,30d時(shí)出現(xiàn)成熟神經(jīng)元標(biāo)記NSE的表達(dá),陽(yáng)性值為29.5%,但細(xì)胞凋亡明顯。鼠腦條件培養(yǎng)基誘導(dǎo)14d時(shí),NSE略有表達(dá),值為12.8%。采用成神經(jīng)球誘導(dǎo)方法進(jìn)行7d成神經(jīng)球誘導(dǎo),再經(jīng)過(guò)7-9天的分化培養(yǎng)。在7d時(shí)有8.9%NSE表達(dá)。β-Ⅲ-Tubulin及Nissl在經(jīng)三種誘導(dǎo)方法誘導(dǎo)成熟后,均為陽(yáng)性表達(dá)。(3)成球法誘導(dǎo)AMSCs向神經(jīng)樣細(xì)胞分化過(guò)程中,取AMSCs及誘導(dǎo)不同時(shí)間點(diǎn)的成神經(jīng)細(xì)胞從細(xì)胞整體及個(gè)體角度分別證實(shí)：在成球誘導(dǎo)法誘導(dǎo)下,通過(guò)7天成神經(jīng)球后,在繼續(xù)的分化過(guò)程1-6天時(shí)細(xì)胞內(nèi)鈣離子濃度先呈上升趨勢(shì),在分化7-9天時(shí)達(dá)到高峰,隨后急劇下落,與細(xì)胞培養(yǎng)時(shí)細(xì)胞發(fā)生凋亡的時(shí)間一致。 結(jié)論：AMSCs較BMSCs具有較高的增殖能力,神經(jīng)球分化方法更能夠高效低損傷的誘導(dǎo)AMSCs分化為類神經(jīng)細(xì)胞,細(xì)胞內(nèi)游離鈣離子在AMSCs向神經(jīng)樣細(xì)胞分化過(guò)程中會(huì)發(fā)生變化,對(duì)維持細(xì)胞分化,存活均起到了一定的作用。
[Abstract]:Objective: to isolate and extract bone marrow mesenchymal stem cells (Bone marrow-derived mesenchymal stem cells, BMSCs) and adipose mesenchymal stem cells (Adipose-derived mesenchymal stem cells, AMSCs) to compare their biological characteristics and proliferation ability in vitro. The effects of three kinds of neural induction methods on the differentiation of AMSCs into neuron-like cells were discussed, and the induction efficiency of AMSCs to neuron-like cells was improved, which laid a foundation for the study of the differentiation mechanism of AMSCs into neuron-like cells. It is helpful to elucidate the mechanism of neurogenesis and differentiation of AMSCs and provide theoretical basis for the application of AMSCs in clinical regenerative medicine. Methods: (1) BMSCs was separated by density gradient centrifugation and AMSCs. was isolated by enzyme digestion in vitro. The surface antigen of MSCs was detected and MSCs was induced by osteogenesis in vitro, and the characteristics of MSCs stem cells were identified by lipogenic induction. (2) three induction schemes induced AMSCs to differentiate into neuron-like cells, including chemical induction, brain conditioned medium induction. Methods of inducing neuronal differentiation. Morphological changes were observed and the expression of 尾-鈪，

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