小鼠星状胶质细胞体外培养及鉴定

小鼠星状胶质细胞体外培养及鉴定(7700字)
摘  要：目的：体外成功分离、培养并鉴定小鼠星状胶质细胞,并建立稳定的培养体系，为进一步制备可供精原干细胞生长的饲养层研究奠定基础。方法：取新生4d昆明系乳鼠，无菌取出脑组织，剥除脑膜、血管结缔组织。剪成乳糜状后，用培养液消化结合机械吹打使组织分散，制成单细胞悬液，过滤离心后得到次细胞悬液，再进行细胞计数。振荡分离纯化后，接种于预先涂有多聚赖氨酸包被的Coning培养瓶中，每3d换液一次。培养10d，待I型星状细胞汇合度达90％以上时进行传代。最后，进行GFAP-FITC免疫荧光法对所获细胞进行鉴定。结果：成功分离、培养并纯化了星状胶质细胞，经GFAP免疫组化鉴定后建立了饲养层。
关键词：星状胶质细胞；原代培养；传代培养；胶质原纤维酸性蛋白

Isolation and characterization of mouse astrocyte
Abstract: Objective: Isolation identification and obtain high purified astrocytes. Establish an affection culture system, which is the base for further preparation for spermatogonial stem cells feeder layer, in vitro. Methods: The neonatal 4 days of mice (Kunming series) were used. Sterile brains were removed, and then stripped meninges, vascular connective tissue. After the cut chyle-like digestion medium combined with mechanical percussion to get organize distributed, to make it into single cell suspension. Filter by sub-cell suspension after centrifugation, then planted into poly L-lysine pre-coated culture flask Coning, after oscillation, separation and purification. The culture media was changed once every 3 days. The cells were passage when it comes to appropriate 90%confluence after 10 days. The cells was identified by rabbit anti mouse GFAP-FITC antibody. Results: We’ve successful separation and identification, high purified astrocytes, after immunohistochemistry identification, we got the feeder layer successfully.
Key words: Astrocyte; Primary culture; Subculture; Glial fibrillary acidic protein;
 

目    录
    摘要……………………………………………………………………………1
    关键词…………………………………………………………………………1
    1前言……………………………………………………………………………2
    2材料与方法……………………………………………………………………3
    2.1材料 ………………………………………………………………………3
    2.1.1实验动物…………………………………………………………………3
    2.1.2仪器设备…………………………………………………………………3
    2.1.3试剂和药品…………………………………………………………………4
    2.2方法…………………………………………………………………………4
    2.2.1星状胶质细胞分离纯化方法……………………………………………4
    2.2.2星状胶质细胞的原代及传代方法………………………………………4
    2.2.3 星状胶质细胞的鉴定方法，GFAP阳性方法……………………………4
    2.2.4星状胶质细胞的传代培养………………………………………………5
    2.2.5星状胶质细胞生长曲线绘制……………………………………………5
    2.2.6星状胶质细胞饲养层制备………………………………………………5
    3 结果…………………………………………………………………………………5
    3.1星状细胞的形态学观察……………………………………………………6
    3.2星状胶质细胞生长曲线………………………………………………………6
    3.3 GFAP鉴定结果………………………………………………………………6
    4 讨论…………………………………………………………………………………6
    5 结论…………………………………………………………………………………8
    参考文献 ……………………………………………………………………………8
    致谢…………………………………………………………………………………9