新生大鼠大脑皮质星形胶质细胞纯化及培养方法的改进
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摘要
目的快速纯化大鼠大脑皮质原代星形胶质细胞并进行传代培养。方法取新生2日龄SD大鼠大脑皮层,每2只为一组,将剥除软脑膜的大脑皮层剪碎,添加0. 25%胰蛋白酶-EDTA溶液500μL,37℃消化15 min,将细胞悬液接种于未用多聚赖氨酸处理过的培养瓶中,37℃、5%CO_2细胞培养箱中孵育15 min后,将细胞以5×10~6个/m L接种于L-多聚赖氨酸包被的T75培养瓶中。待细胞长满瓶底,200 r/min 37℃恒温摇18 h,加入1 m L 0. 25%胰蛋白酶,待大部分细胞悬浮后收集细胞。在倒置相差显微镜下对细胞进行形态学观察,胶质纤维酸性蛋白免疫荧光染色法进行星形胶质细胞纯度鉴定,并以MTS法测定传代后细胞增殖活力。结果星形胶质细胞纯度达到(97. 86±0. 91)%,细胞传代后生长情况及增殖活力良好。结论该方法可以高效获取原代及传代的大鼠大脑皮质星形胶质细胞。
OBJECTIVE To establish an effective method of primary and passage cultured cerebral cortical astrocyte of SD neonatal rat in vitro. METHODS Cerebral cortex of two 2-day-old SD rats were taken with aseptic operation and then were cut to pieces. After stripped the pia mater,digested by 500 μL 0. 25% trypsin at 37 ℃ for 15 min. Next,dispersed cell suspension was made by mechanical method and filtered. Cell suspensions were incubated in an uncoated culture bottle at 37 ℃ for 15 min. The cells were inoculated at 5×10~6/m L in the T75 culture flask coated with L-polylysine. The cells were shaken at 200 r/min 37 ℃ for 18 h,then added 1 m L of trypsin to digest cells and then collected the cells. The morphology of the passage cells was observed under inverted phase contrast microscope, and the purity of astrocytes was identified by immunofluorescence staining of GFAP. The proliferate activity of passage cells was determined by MTS assay. RESULTS The purity of astrocytes was( 97. 86 ± 0. 91) %,and the growth and proliferation activity of astrocytes were good after passage.CONCLUSION A rapid,economical and effective method for obtaining astrocytes in the cerebral cortex of newborn rats was established.
OBJECTIVE To establish an effective method of primary and passage cultured cerebral cortical astrocyte of SD neonatal rat in vitro. METHODS Cerebral cortex of two 2-day-old SD rats were taken with aseptic operation and then were cut to pieces. After stripped the pia mater,digested by 500 μL 0. 25% trypsin at 37 ℃ for 15 min. Next,dispersed cell suspension was made by mechanical method and filtered. Cell suspensions were incubated in an uncoated culture bottle at 37 ℃ for 15 min. The cells were inoculated at 5×10~6/m L in the T75 culture flask coated with L-polylysine. The cells were shaken at 200 r/min 37 ℃ for 18 h,then added 1 m L of trypsin to digest cells and then collected the cells. The morphology of the passage cells was observed under inverted phase contrast microscope, and the purity of astrocytes was identified by immunofluorescence staining of GFAP. The proliferate activity of passage cells was determined by MTS assay. RESULTS The purity of astrocytes was( 97. 86 ± 0. 91) %,and the growth and proliferation activity of astrocytes were good after passage.CONCLUSION A rapid,economical and effective method for obtaining astrocytes in the cerebral cortex of newborn rats was established.
引文
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[2]McKENNA M C,STRIDH M H,McNAIR L F,et al.Glutamate oxidation in astrocytes:roles of glutamate dehydrogenase and aminotransferases[J].Neurosci Res,2016,94:1561-1571．
[3]LEE J Y,JOO B,NAM J H,et al.An aqueous extract of herbal medicine ALWPs enhances cognitive performance and inhibits LPS-induced neuroinflammation via FAK/NF-κB signaling pathways[J].Front Aging Neurosci,2018,10:269．
[4]MAO X,LIU J,CHEN C,et al.PCBP2 modulates neural apoptosis and astrocyte proliferation after spinal cord injury[J].Neurochem Res,2016;41(9):2401-2414．
[5]丁娟，丁敬宾，马小虎，等．一种改进的大鼠大脑皮质星形胶质细胞的培养方法[J]．神经解剖学杂志，2017,33(3):349-353
[6]王建斌，冯敏，廖芸，等．恒河猴大脑皮质星形胶质细胞的原代培养及鉴定[J]中国细胞生物学学报，2017,39(1):58-64．
[7]LI S,UNO Y,RUDOLPH U,et al.Astrocytes in primary cultures express serine racemase,synthesize d-serine and acquire A1 reactive astrocyte features[J].Biochem Pharmacol,2018,151:245-251．
[8]NAM Y,KIM J H,KIM J H,et al.Reversible Induction of pain hypersensitivity following optogenetic stimulation of spinal astrocytes[J].Cell Rep,2016,17(11):3049-3061．
[9]KOSS K M,CHURCHWARD M A,JEFFERY A F,et al.Improved 3D hydrogel cultures of primary glial cells for in vitro modelling of neuroinflammation[J].Jo VE,2017(130):e56615．
[10]WEI Z,KALE S,RABINOVSKY R,et al.Cocultures of glioma stem cells and primary neurons,astrocytes,microglia,and endothelial cells for investigation of intercellular communication in the brain[J].Front Neurosci,2019,13:361.