本文選題：骨髓間充質(zhì)干細(xì)胞(bone + marrow��； 參考：《大連醫(yī)科大學(xué)》2010年碩士論文

【摘要】： 目的：神經(jīng)系統(tǒng)疾病如脊髓損傷、退行性病、多發(fā)硬化、運(yùn)動(dòng)神經(jīng)元病等,主要原因是神經(jīng)細(xì)胞的壞死、功能的缺失。干細(xì)胞(stem cells)是一類具有無限自我更新和多向分化潛能的細(xì)胞,在一定條件下,可分化為神經(jīng)元樣細(xì)胞和神經(jīng)膠質(zhì)樣細(xì)胞,有望作為神經(jīng)系統(tǒng)疾病細(xì)胞移植的種子細(xì)胞,但是干細(xì)胞種類繁多,據(jù)其發(fā)生學(xué)來源的不同可分為胚胎干細(xì)胞(embryonic stem cells, ESCs)和成體干細(xì)胞(adult stem cells, ASCs),其中ASCs是指存在于已分化組織的未分化細(xì)胞。ASCs較ESCs的優(yōu)點(diǎn)是取材相對(duì)方便,不存在倫理學(xué)問題。目前研究較多的成體干細(xì)胞主要是骨髓間充質(zhì)干細(xì)胞(bone marrow mesenchymal stem cells, BMSCs),它取材方便,易于分離純化,神經(jīng)向分化能力的研究也比較多,已應(yīng)用于前期臨床研究試驗(yàn),而脂肪間充質(zhì)干細(xì)胞(Adipose-derived Mesenchymal Stem Cells,ADSCs)作為近年來新發(fā)現(xiàn)的種子細(xì)胞,神經(jīng)向分化能力的研究還相對(duì)較少,但它具有BMSCs所不具備的優(yōu)點(diǎn),如：獲取更為方便,對(duì)人體創(chuàng)傷小,很少受血液及免疫系統(tǒng)的影響等。本實(shí)驗(yàn)旨在比較人類BMSCs與ADSCs的神經(jīng)向分化能力,為干細(xì)胞移植治療神經(jīng)系統(tǒng)疾病篩選理想種子細(xì)胞。 方法：1)采用Percoll分離法在體外培養(yǎng)及擴(kuò)增BMSCs,利用胰酶消化的方法獲取ADSCs。通過表面抗原檢測(cè)的方法進(jìn)行鑒定。2)采用多種生長因子聯(lián)合誘導(dǎo)方案誘導(dǎo)ADSCs、BMSCs神經(jīng)向分化,即先用濃度為10 ng/ml的bFGF預(yù)誘導(dǎo)24 h,再加入20 ng/ml的bFGF、20 ng/ml的EGF、20 ng/ml的BDNF,分別于24 h、3 d、7 d、10 d、14 d時(shí)進(jìn)行免疫熒光檢測(cè)Nestin,β-Ⅲ-Tubulin、NSE的表達(dá),尼氏熒光染色檢測(cè)尼氏小體表達(dá),觀察細(xì)胞形態(tài)變化。3)統(tǒng)計(jì)陽性細(xì)胞的比率,卡方檢驗(yàn)分析統(tǒng)計(jì)結(jié)果。 結(jié)果：1.采用percoll密度梯度離心法成功分離出人骨髓間充質(zhì)干細(xì)胞,細(xì)胞表型鑒定為CD90陽性,CD106陽性,CD34陰性。2.通過對(duì)比ADSCs和BMSCs的生長曲線發(fā)現(xiàn),脂肪間充質(zhì)干細(xì)胞較骨髓間充質(zhì)干細(xì)胞具有更強(qiáng)的增殖能力。3.利用生長因子聯(lián)合誘導(dǎo)的方式誘導(dǎo)BMSCs和ADSCs神經(jīng)向分化,誘導(dǎo)后,BMSCs和ADSCs細(xì)胞形態(tài)都發(fā)生了改變,胞體變圓并伸出細(xì)長突起,對(duì)于分化細(xì)胞的神經(jīng)細(xì)胞特異標(biāo)記的鑒定也具有相同的趨勢(shì)：誘導(dǎo)中期Nestin、β-Ⅲ-tublin顯著增多,晚期稍降,而NSE和Nissl小體誘導(dǎo)晚期才出現(xiàn)具有相似的陽性率。 結(jié)論：1.ADSCs具有較BMSCs更為活躍的生長、增殖能力；2.ADSCs具有與BMSCs類似的神經(jīng)向分化能力。
[Abstract]:Objective: neurologic diseases such as spinal cord injury, degenerative disease, multiple sclerosis, motor neuron disease, etc. Stem cells are a kind of cells with infinite self-renewal and multi-differentiation potential. Under certain conditions, stem cells can be differentiated into neuron-like cells and glial cells, which may be used as seed cells for transplantation of cells in nervous system diseases. However, stem cells can be divided into embryonic stem cells (ESCs) and adult stem cells (adult stem cells) according to the different origin of stem cells. ASCs refers to undifferentiated cells that exist in differentiated tissues. ASCs are more convenient than ESCs. There is no ethical problem. At present, the main adult stem cells studied are bone marrow mesenchymal stem cells (BMSCs), bone marrow mesenchymal stem cells (BMSCs). As a newly discovered seed cell in recent years, adipose mesenchymal stem cells (Adipose-derived Mesenchymal Stem cells ADSCs) have relatively little research on the ability of neural differentiation, but it has the advantages that BMSCs does not. For example, it is more convenient to obtain and less invasive to human body. Rarely affected by the blood and immune system, etc. The purpose of this study was to compare the neural differentiation ability of human BMSCs and ADSCs and to screen ideal seed cells for stem cell transplantation in the treatment of nervous system diseases. Methods BMSCs were cultured and amplified by Percoll in vitro, and ADSCs were obtained by trypsin digestion. The neural differentiation of ADSCsBMSCs was induced by a combination of multiple growth factors and the detection of surface antigen. The expression of Nestin, 尾-鈪，

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