本文關(guān)鍵詞：膠質(zhì)細(xì)胞源性神經(jīng)營養(yǎng)因子體外誘導(dǎo)骨髓間充質(zhì)干細(xì)胞向神經(jīng)細(xì)胞分化的研究　出處：《揚(yáng)州大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 骨髓間充質(zhì)干細(xì)胞 膠質(zhì)細(xì)胞源性神經(jīng)營養(yǎng)因子 誘導(dǎo) 分化

【摘要】：目的：探討體外分離、培養(yǎng)大鼠骨髓間充質(zhì)干細(xì)胞(MSCs)的方法,證實(shí)提純的骨髓間充質(zhì)干細(xì)胞與膠質(zhì)細(xì)胞源性神經(jīng)營養(yǎng)因子(GDNF)共培養(yǎng),可定向誘導(dǎo)分化為神經(jīng)細(xì)胞。 方法：1.選用健康SD大鼠,采用全骨髓貼壁法體外培養(yǎng)、純化MSCs。2.采用流式細(xì)胞術(shù)進(jìn)行細(xì)胞表型鑒定,測定CD34、CD44、CD45、CD90的陽性細(xì)胞百分率。3.分別采用對照組、GDNF組對MSCs進(jìn)行誘導(dǎo)分化研究。4.采用倒置相差顯微鏡連續(xù)觀察細(xì)胞生長情況及形態(tài)變化。采用免疫組化法檢測神經(jīng)前體細(xì)胞的特異性標(biāo)志神經(jīng)巢蛋白(Nestin),神經(jīng)細(xì)胞的特異性標(biāo)志神經(jīng)元特異性烯醇化酶(neon-specific enolase, NSE)。 結(jié)果：原代提取獲得的骨髓間充質(zhì)干細(xì)胞呈梭形,類似于成纖維細(xì)胞,培養(yǎng)第4代的MSCs形態(tài)均一性較好,經(jīng)流式細(xì)胞儀檢測純度較高,四種細(xì)胞表型的陽性率分別是：CD34:3.98%, CD44:98.74%, CD45:1.03%, D90:98.08%。MSCs經(jīng)GDNF誘導(dǎo)后,細(xì)胞胞體逐漸向胞核收縮,變形細(xì)胞逐漸增多,出現(xiàn)雙極、多極和錐形典型的神經(jīng)細(xì)胞形態(tài),細(xì)胞邊緣出現(xiàn)突起,且與鄰近細(xì)胞突起逐漸相互連接。GDNF組分別于誘導(dǎo)后6h出現(xiàn)nestin表達(dá)陽性,24h后誘導(dǎo)達(dá)頂峰。12h檢測到神經(jīng)細(xì)胞的特異性標(biāo)志NSE表達(dá)陽性,且表達(dá)強(qiáng)度隨時(shí)間逐漸增加,48h后誘導(dǎo)達(dá)頂峰。72h觀察到誘導(dǎo)細(xì)胞逐漸凋亡脫落。陰性對照組未出現(xiàn)神經(jīng)細(xì)胞的形態(tài)變化及特異性標(biāo)志的陽性表達(dá)。 結(jié)論：大鼠骨髓間充質(zhì)干細(xì)胞可通過全骨髓貼壁法在體外進(jìn)行原代提取分離及傳代培養(yǎng),所獲得細(xì)胞生長穩(wěn)定、增殖迅速,經(jīng)GDNF誘導(dǎo)后具有向神經(jīng)元樣細(xì)胞分化的潛能。
[Abstract]:Objective: To explore the method of isolation and culture of rat bone marrow mesenchymal stem cells (MSCs) in vitro. It is confirmed that purified bone marrow mesenchymal stem cells and glial cell line derived neurotrophic factor (GDNF) co cultured, can be induced to differentiate into neural cells.
Methods: 1. healthy SD rats by whole bone marrow adherent culture, purification of MSCs.2. by phenotype by flow cytometry, the determination of CD34, CD44, CD45, CD90 and.3. respectively by the percentage of positive cells in control group, GDNF group of MSCs induced differentiation of.4. by inverted phase contrast microscope observation the cell growth and morphological changes. The immune group specific marker nestin was detected in neural precursor cells (Nestin), a specific marker of neuron specific enolase in nerve cells (neon-specific enolase, NSE).
Results: the primary extraction of bone marrow mesenchymal stem cells were spindle shaped, similar to fibroblast cells, cultured MSCs morphological homogeneity of the fourth generation of good, high purity was detected by flow cytometry, the positive rate of four kinds of cell phenotypes were CD34:3.98%, CD44:98.74%, CD45: 1.03%, D90:98.08%.MSCs induced by GDNF the cell body, gradually to nucleus shrinkage, deformation of cells gradually increased, appeared bipolar, multipolar and typical conical nerve cell morphology, cell edge projection, and the adjacent cell processes gradually connected with each other in group.GDNF were induced after 6h nestin positive expression reached the peak detected by.12h nerve cells induced by 24h after specific marker NSE expression, and the expression increased gradually with time, reached a peak at 48h after induction of.72h induced cell apoptosis observed gradually fall off. The negative control group did not show morphological changes of nerve cells. Positive expression of specific markers.
Conclusion: rat bone marrow mesenchymal stem cells can be extracted, separated and cultured in vitro by whole bone marrow adherent method. The cells obtained are stable and proliferated rapidly. After GDNF induction, they have the potential to differentiate into neuron like cells.

【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R329.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前5條

1 胡繼良;姜曉丹;鄒雨汐;郭燕舞;周德祥;徐如祥;;成年小鼠腦室下區(qū)神經(jīng)干細(xì)胞培養(yǎng)與鑒定體系的建立[J];南方醫(yī)科大學(xué)學(xué)報(bào);2008年11期

2 邢瑩;白瑞櫻;鄢文海;韓雪飛;段萍;許燕;樊志剛;;人骨髓間充質(zhì)干細(xì)胞向神經(jīng)細(xì)胞分化過程中Notch通路信號分子表達(dá)的變化[J];生理學(xué)報(bào);2007年03期

3 王新生;崔慧先;劉華;孫黎;;黃芪誘導(dǎo)骨髓間充質(zhì)干細(xì)胞分化進(jìn)程中細(xì)胞內(nèi)鈣離子濃度的動(dòng)態(tài)變化[J];中國組織工程研究與臨床康復(fù);2007年42期

4 裴晶晶;吳潤;趙紅斌;劉旭東;胡軍;白孟海;;Ca~(2+)信號介導(dǎo)紅景天苷促進(jìn)小鼠骨髓間充質(zhì)干細(xì)胞向神經(jīng)細(xì)胞的定向分化[J];中國組織工程研究與臨床康復(fù);2010年10期

5 王小梅;穆長征;馬云勝;;Wnt3a促進(jìn)大鼠骨髓間充質(zhì)干細(xì)胞向神經(jīng)元樣細(xì)胞的分化(英文)[J];中國組織工程研究與臨床康復(fù);2010年23期

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