大鼠脂肪干细胞向神经样细胞分化的研究

发布时间：2018-08-17 11:40

【摘要】：目的探索并固定大鼠脂肪干细胞获得、增殖和传代的方法和操作流程，并且通过诱导脂肪干细胞分化为成脂细胞、成骨细胞而证明脂肪干细胞的多向分化能力。方法取质量约120克的SD大鼠2只，性别不限，分离其腹股沟脂肪组织，将其剪碎并用滤网过滤，然后经一系列操作而将含有脂肪干细胞的培养液平铺于25cm2培养瓶中培养，并进而增殖、传代。之后，通过细胞流式分析检测第三代细胞表型证明其为脂肪干细胞。之后分别更换成脂诱导培养基和成骨细胞培养基，诱导脂肪干细胞定向分化为脂肪细胞和骨细胞。结果成功分离培养出脂肪干细胞，并能进行增殖和传代。利用流式细胞技术检测脂肪干细胞的CD34(+)、CD44（+）、CD45(-)、CD106（+），证明该细胞为脂肪干细胞。而向脂肪和成骨细胞诱导的细胞均发生了明显变化，最后分别通过油红O染色和碱性磷酸酶试剂盒证明脂肪干细胞分化为脂肪细胞和成骨细胞。结论脂肪组织中存在脂肪干细胞，并能够成功的分离、增殖和传代。并且拥有多向分化能力。可以成功的诱导分化为脂肪细胞和成骨细胞。目的：使用神经细胞诱导液将脂肪间充质干细胞定向诱导分化为神经样细胞。方法：首先是将第三代脂肪间充质干细胞在培养瓶中贴壁达到80%以上时消化，然后使其重新贴壁于六孔板中，细胞数量约为105级。然后用添加了bFGF和EGF以及B27的Neurobasal培养基培养六孔板中的细胞。每3天半量添加新的培养液，至5-7天左右就可以看到细胞增殖形成培养液中悬浮的细胞球。取诱导培养第7天的细胞球进行nestin的免疫荧光染色。最后，将培养好的细胞球吹散，离心，并将之培养在仅添加了B27的Neurobasal培养基中诱导分化10天，然后再进行NeuN、GFAP和β-tubulin荧光染色来鉴定分化成的细胞。结果：在使用了含有bFGF和EGF以及B27的Neurobasal培养基诱导了5天时就出现了悬浮的细胞球，并且通过免疫荧光染色证明为nestin阳性，证明其为神经干细胞的细胞球。而在使用了仅添加了B27的Neurobasal培养基中诱导分化10天后，打散的细胞球细胞也分化成了神经细胞，并经过了MAP2ab、NeuN、GFAP和β-tubulin荧光染色鉴定，证明分化成了神经元和星形胶质细胞。结论：经特定的诱导培养液诱导后，，脂肪间充质干细胞可以被诱导分化为神经干细胞并且进一步分化为神经元和胶质细胞。
[Abstract]:Objective to explore and immobilize the methods and procedures of obtaining, proliferating and passaging adipose stem cells in rats, and to prove the ability of adipose stem cells to differentiate into adipocytes and osteoblasts by inducing adipose stem cells to differentiate into adipogenic cells and osteoblasts. Methods two SD rats of about 120g mass, with unlimited sex, were isolated from their inguinal adipose tissue, then shredded and filtered by filter, and then cultured in 25cm2 culture flask with a series of culture medium containing adipose stem cells. And then proliferate, subculture. After that, the third generation cells were identified as adipose stem cells by flow cytometry. Then the adipogenic medium and osteoblast medium were changed to induce adipocytes to differentiate into adipocytes and osteoblasts. Results Adipose stem cells were successfully isolated and cultured, and could be proliferated and subcultured. CD34 () CD44 (-) CD45 (-) CD106 (), of adipose stem cells was detected by flow cytometry. The differentiation of adipocytes into adipocytes and osteoblasts was proved by oil red O staining and alkaline phosphatase kit. Conclusion adipose stem cells exist in adipose tissue and can be successfully isolated, proliferated and subcultured. And has the ability to differentiate in multiple directions. Adipocytes and osteoblasts can be successfully induced to differentiate into adipocytes and osteoblasts. Aim: to differentiate adipose mesenchymal stem cells into neuronal like cells by neuronal induction. Methods: first, the third generation adipose mesenchymal stem cells were digested when the adherent reached 80% in the culture bottle, and then the cells were reattached to the six-hole plate. The number of cells was about 105 grade. Then the cells in the six-well plate were cultured on Neurobasal medium supplemented with bFGF and EGF and B 27. After adding the new medium every 3 and a half days, the cell proliferation can be seen in the culture medium from 5 to 7 days, and the cell ball suspended in the culture medium can be seen. The cells were cultured on the 7th day after induction for nestin immunofluorescence staining. Finally, the cultured cells were blown out, centrifuged, and cultured in Neurobasal medium supplemented with only B27 for 10 days, then the differentiated cells were identified by Neun GFAP and 尾 -tubulin fluorescence staining. Results: after 5 days of induction with Neurobasal medium containing bFGF, EGF and B27, suspended cell spheres were found, and were proved to be nestin positive by immunofluorescence staining, which were proved to be neural stem cells. On the other hand, after 10 days of differentiation induced by Neurobasal medium supplemented with only B27, the dispersed cells were also differentiated into neurons, and identified by MAP2ababa NeuNu GFAP and 尾 -tubulin fluorescence staining, it was proved that the cells were differentiated into neurons and astrocytes. Conclusion: adipose mesenchymal stem cells can be induced to differentiate into neural stem cells and further differentiate into neurons and glial cells.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329.2

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