大鼠大腦皮層星形膠質(zhì)細(xì)胞體外培養(yǎng)改良方法的研究

閱讀次數(shù)：669 發(fā)布時(shí)間：2018/2/6 10:15:49

A modified culture method for astrocytes from rat cortical tissue in vitro

OBJECTIVE: To evaluate the efficiency of a modified culture method for rat cerebral cortical astrocytes in vitro. METHODS: The astrocytes derived from the cerebral cortex of 3-day-old Sprague-Dawley rats were first purified as described previously, then the cells were replanted at a low density. The culture flask was changed after 1 hour and substratum was replaced after 24 hours. Cells were syncretized to a monolayer, followed by cell passage. After three passages the cells were cultured in DMEM medium containing 10% fetal serum for a long period. The derivation of the cells was identified by immunofluorescent staining with anti-GFAP polyclonal antibodies. RESULTS: A variety of morphologically distinct astrocytes with many long processes and small cell bodies were obtained. Finally an astrocytic network occurred through cellular process connections. The immunofluorescent staining demonstrated the percentage of GFAP-positive cells was above 98%. CONCLUSIONS: The modified culture method for astrocytes from rat cerebral tissue is reliable, with a high purity. The cultured astrocytes have a similar morphological development to those in vivo.

大鼠大腦皮層星形膠質(zhì)細(xì)胞體外培養(yǎng)改良方法的研究

目的：改進(jìn)大鼠大腦皮質(zhì)星形膠質(zhì)細(xì)胞（astrocyte，AS）的體外培養(yǎng)方法，為進(jìn)一步研究奠定基礎(chǔ)。方法：選取生后3 d的 Sprague-Dawley大鼠的大腦皮層，常規(guī)分離純化AS，低密度種植，培養(yǎng)箱中孵育 1 h后換瓶，24 h后換液，細(xì)胞融合成單層后傳代，傳代后維持在含10%胎牛血清DMEM培養(yǎng)基中培養(yǎng)，并在長(zhǎng)時(shí)期內(nèi)不給予更換或補(bǔ)加培養(yǎng)液。采用 GFAP/DAPI免疫熒光法鑒定AS細(xì)胞。結(jié)果：體外培養(yǎng)的純化大鼠大腦皮層AS，表現(xiàn)為細(xì)胞突起細(xì)長(zhǎng)、胞體明顯縮小、形態(tài)多樣的纖維型AS，最后細(xì)胞突起之間相互連接形成AS網(wǎng)絡(luò)。GFAP/DAPI 免疫熒光染色結(jié)果示GFAP陽(yáng)性細(xì)胞達(dá) 98%以上。結(jié)論：大鼠大腦皮質(zhì)AS的體外培養(yǎng)改良方法培養(yǎng)的AS體外形態(tài)、發(fā)育特點(diǎn)均與在體內(nèi)AS基本一致，且純度較高，為進(jìn)一步進(jìn)行AS的研究創(chuàng)造了條件。

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