本文選題：骨髓間充質(zhì)干細(xì)胞 + 分離培養(yǎng)　； 參考：《湖北中醫(yī)學(xué)院》2009年碩士論文

【摘要】： 目的: 1.探討大鼠骨髓間充質(zhì)干細(xì)胞(mesenchymal stem cells,MSCs)的分離培養(yǎng)和體外擴(kuò)增方法。 2.研究其在當(dāng)歸注射液或β-巰基乙醇(β-Mercaptoethanol,BME)誘導(dǎo)劑誘導(dǎo)下向神經(jīng)元細(xì)胞分化的能力,以提供一種較好的誘導(dǎo)方案。 3.明確堿性成纖維生長因子(Basic fibroblast growth factor,bFGF)在誘導(dǎo)劑定向誘導(dǎo)過程中所起的作用,以優(yōu)化誘導(dǎo)方案。 4.觀察大鼠骨髓間充質(zhì)干細(xì)胞的分離培養(yǎng)、體外擴(kuò)增及定向誘導(dǎo)分化為神經(jīng)元樣細(xì)胞過程中的生物學(xué)特性,為今后臨床研究提供良好的種子細(xì)胞來源。 方法: 無菌條件下取大鼠骨髓,采用全骨髓貼壁培養(yǎng)篩選法分離出成年大鼠MSCs,進(jìn)行體外培養(yǎng)、擴(kuò)增,觀察其生物學(xué)特性。取生長良好的第5代MSCs經(jīng)堿性成纖維生長因子(bFGF)預(yù)誘導(dǎo)后用當(dāng)歸注射液或β-巰基乙醇(β-Mercaptoethanol,BME)誘導(dǎo)MSCs向神經(jīng)元樣細(xì)胞分化,并針對(duì)堿性成纖維生長因子(bFGF)在誘導(dǎo)過程所起作用設(shè)立三組實(shí)驗(yàn)對(duì)照組,包括對(duì)照組1:預(yù)誘導(dǎo)液中不含bFGF和誘導(dǎo)液中不含當(dāng)歸注射液或BME誘導(dǎo)劑;對(duì)照組2:預(yù)誘導(dǎo)液中含bFGF,而誘導(dǎo)液中不含當(dāng)歸注射液或BME誘導(dǎo)劑;對(duì)照組3:預(yù)誘導(dǎo)液中不含bFGF,而誘導(dǎo)液中含有當(dāng)歸注射液誘導(dǎo)劑。倒置顯微鏡下觀察誘導(dǎo)過程中細(xì)胞形態(tài)所發(fā)生的變化,并通過細(xì)胞免疫化學(xué)法鑒定經(jīng)各組誘導(dǎo)后所得細(xì)胞的神經(jīng)干細(xì)胞標(biāo)志物神經(jīng)巢蛋白(neuroepithelial stem cell protein,Nestin)、神經(jīng)元特異性烯醇化酶(Neuron-specific Enolase,NSE)、膠質(zhì)纖維酸性蛋白(glialfibrillary acidic protein,GFAP)的表達(dá)。經(jīng)鏡下計(jì)數(shù)誘導(dǎo)分化后所得細(xì)胞的陽性表達(dá)率,用統(tǒng)計(jì)學(xué)q檢驗(yàn)方法比較各組之間的差異。 結(jié)果: 大鼠MSCs可用貼壁培養(yǎng)篩選法成功分離并在體外大量擴(kuò)增。經(jīng)當(dāng)歸注射液或BME誘導(dǎo),MSCs可向神經(jīng)元樣細(xì)胞分化,均出現(xiàn)胞體和突起,細(xì)胞免疫化學(xué)染色神經(jīng)元特異性烯醇化酶(NSE),巢蛋白(Nestin)陽性,神經(jīng)膠質(zhì)纖維酸性蛋白(GFAP)呈陰性。且經(jīng)堿性成纖維生長因子(bFGF)預(yù)誘導(dǎo)的當(dāng)歸組較未經(jīng)bFGF預(yù)誘導(dǎo)的對(duì)照組3誘導(dǎo)成神經(jīng)元樣細(xì)胞的陽性細(xì)胞表達(dá)率明顯提高。 結(jié)論: 貼壁分離培養(yǎng)法分離的MSCs能在體外培養(yǎng)條件下生長良好、可連續(xù)傳代,體外培養(yǎng)擴(kuò)增能力較強(qiáng),以當(dāng)歸注射液或β-巰基乙醇作用于MSCs可成功誘導(dǎo)出神經(jīng)元樣細(xì)胞,MSCs能夠成為理想的種子細(xì)胞來源。且堿性成纖維生長因子(bFGF)在誘導(dǎo)過程中起積極作用。
[Abstract]:Objective: 1. To investigate the isolation, culture and in vitro amplification of mesenchymal stem cells from rat bone marrow mesenchymal stem cells. 2. To study its ability to differentiate into neuron cells induced by Angelica sinensis injection or 尾 -Mercaptoethanolanol (BME) inducer, to provide a better induction scheme. 3. The role of basic fibroblast growth factor bFGFin in the induction of basic fibroblast growth factor (BFGF) was determined in order to optimize the induction scheme. 4. To observe the biological characteristics of rat bone marrow mesenchymal stem cells (BMSCs) in vitro amplification and differentiation into neuron-like cells and to provide a good source of seed cells for future clinical research. Methods: Adult rat MSCs were isolated from rat bone marrow in aseptic condition by whole bone marrow adherent culture screening method, and then cultured in vitro and amplified, and their biological characteristics were observed. The fifth passage of MSCs with good growth was preinduced by basic fibroblast growth factor (bFGF). MSCs was induced to differentiate into neuron-like cells by Angelica sinensis injection or 尾 -Mercaptoethanolol (尾 -Mercaptoethanol). According to the effect of basic fibroblast growth factor (bFGF) on the induction process, three experimental control groups were established, including control group 1: no bFGF in the preinduction solution and no angelica injection or BME inducer in the induced solution; In the control group, bFGF2 was found in the preinduction solution, but no angelica injection or BME inducer was found in the induced solution, while in the control group bFGF3 was not found in the preinducible solution, and the inducer in the induced solution was angelica sinensis injection. The changes of cell morphology during induction were observed under inverted microscope. The expression of Nestinan, Neuron-specific Enolase NSEE, glial fibrillary acidic protein (GFAPs) and neuron-specific enolase (Neuron-specific EnolaseNSEP) were detected by immunocytochemistry. The positive expression rate of the cells induced by differentiation was counted under microscope, and the difference between the groups was compared with the method of statistical Q test. Results: Rat MSCs was successfully isolated by adherent culture and expanded in vitro. BME could differentiate into neuron-like cells after Angelica sinensis injection or BME. The neuronal bodies and processes were found. The neuron-specific enolase (NSE) and nestin (nestin) were positive and glial fibrillary acidic protein (GFAP) was negative by immunocytochemistry. The expression rate of neuron-like cells in Angelica sinensis group preinduced by basic fibroblast growth factor (bFGF) was significantly higher than that in control group 3 without bFGF. Conclusion: The MSCs isolated by adherent culture method can grow well in vitro and can be subcultured continuously. MSCs treated with angelica sinensis injection or 尾 -mercaptoethanol could be used as an ideal seed cell source. Basic fibroblast growth factor (bFGF) plays an active role in the induction process.
【學(xué)位授予單位】：湖北中醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R329

【引證文獻(xiàn)】

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本文編號(hào)：1848837