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;
[资料来源:http://www.doc163.com]
目 录
摘要……………………………………………………………………………1
关键词…………………………………………………………………………1
1前言……………………………………………………………………………2 [资料来源:http://Doc163.com]
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 [来源:http://Doc163.com]
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
[资料来源:http://www.doc163.com]
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 [资料来源:Doc163.com]
4 讨论…………………………………………………………………………………6
5 结论…………………………………………………………………………………8
参考文献 ……………………………………………………………………………8
致谢…………………………………………………………………………………9
[资料来源:http://www.doc163.com]