SD大鼠脑皮层星形胶质细胞的原代培养及纯化方法改良

The improvement on the primary culture and the purification of astrocytes from cerebral cortex of SD rats

目的:探讨大鼠大脑皮层星形胶质细胞的培养方法,以获得纯度高的细胞进一步对其进行体外实验研究。方法:在Mc-Carthy方法基础上改用出生后1d的SD大鼠的大脑皮层,结合机械分离和胰酶消化后制备单细胞悬液接种,差速贴壁1h,培养14d以后上摇床以去除混杂细胞,细胞融合后传代,每次传代前都经历胰酶冲洗和差速贴壁,传3代后利用GFAP-FITC免疫荧光法对所获细胞进行鉴定。结果:经过原代传代及纯化,大脑皮层星形胶质细胞体外培养成功,并有较高的纯度。GFAP-FITC免疫荧光染色阳性。结论:经过合理取材、改良纯化方法后,可以更为简便得到纯度很高的星形胶质细胞。

Objective： To improve the method of astrocytes culture and in order to get rich astrocytes from SD rat cerebral cortex which could be utilized in vitro experiments. Methods： We amend the method of McCarthy＇s. The neonatal rat cerebral cortex was made into suspension by mechanical and trypsin dissociation and digestion, and then reduced other cells by differential velocity adherent technique, shaking in orbital shaker after 14 day＇s cell culture, passage cells after cell fusion. Trypsin washing and differential velocity adherent technique were used before each transfer of culture. After subculture and purification, the cultured ceils were identified by immunofluorescence stained with anti-GFAP antibody. Results： After primary cell culture and purification, we got successfully high parity astrocytes. GFAP - F ITC immunofluorescent staining for positive. Conclusion.. After reasonable selection of animal and improvement of purification, we can simply get high purity astrocytes.