一种改进的星形胶质细胞原代培养方法
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摘要
目的改进大鼠大脑皮质星形胶质细胞的体外培养,旨在通过较简便的方法获得高纯度形态典型的星形胶质细胞,为研究星形胶质细胞的生物学作用提供实验模型。方法以新生大鼠为星形胶质细胞来源,利用不同规格的吸头相互叠套,逐步机械吹打,使细胞分散,制备单细胞悬液;获得的单细胞悬液用差速黏附处理去除成纤维细胞,原代培养14d后,恒温振荡法去除小胶质细胞等杂质细胞;胶质细胞原纤维酸性蛋白(GFAP)和S100β免疫荧光共染色,鉴定细胞纯度。结果这种改进的星形胶质细胞培养方法 ,分离培养出星形胶质细胞,阳性率达95%以上。结论采用机械吹打方法建立原代星形胶质细胞培养体系,是一种比较简便、值得推广的可用于体外细胞培养研究工作的有效方法。
Objective The purpose is to introduce a simplified but effective method for astrocytes culture. Method Newborn Sprague-Dawley(SD) rats(1- day old) were sacrificed by decapitation. The single cell suspension of the cortex of cerebrum was prepared by repeated mechanical pipetting. Differential attachment was used to remove contaminating fibroblasts in the cell suspension. The remaining cells were cultured for 14 days, then were agitated on the rotary shaker(180 rpm, 37 C) to remove microglia. The cells were identified as astrocytes by immunofluorescence staining with anti-GFAP antibody and anti-S100β antibody. Results The rat astrocyte was successfully isolated and the proportion of astrocyte was> 95%. Conclusion This modified cultivation method of astrocyte from rat cerebral tissue is established with high purity, and in a good growth condition.
Objective The purpose is to introduce a simplified but effective method for astrocytes culture. Method Newborn Sprague-Dawley(SD) rats(1- day old) were sacrificed by decapitation. The single cell suspension of the cortex of cerebrum was prepared by repeated mechanical pipetting. Differential attachment was used to remove contaminating fibroblasts in the cell suspension. The remaining cells were cultured for 14 days, then were agitated on the rotary shaker(180 rpm, 37 C) to remove microglia. The cells were identified as astrocytes by immunofluorescence staining with anti-GFAP antibody and anti-S100β antibody. Results The rat astrocyte was successfully isolated and the proportion of astrocyte was> 95%. Conclusion This modified cultivation method of astrocyte from rat cerebral tissue is established with high purity, and in a good growth condition.
引文
[1]Chen Y,Swanson RA.Astrocytes and brain injury[J].Cereb Blood Flow Metab,2003,23(2):137-149.
[2]薛庆善,郭宛华.大鼠大脑皮质星形胶质细胞的体外培养及其神经突起生长作用研究[J].神经解剖学杂志,1996,12(2):151-155.
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[6]Rothermundt M,Peters M,Prehn JH,et al.S100B in brain damage and neurodegeneration[J].Microsc Res Tech,2003,60(6):614-632.
[7]Sofroniew MV,Vinters HV.Astrocytes:biology and pathology[J].Acta Neuropathol,2010,119(1):7-35.
[8]张彤,张祥建,刘瑞春,等.胶质纤维酸性蛋白与中枢神经系统疾病[J].脑与神经疾病杂志,2005,13(6):466.
[9]吴雨虹,王慧君,王欣.大鼠原发性脑干损伤后S100B和GFAP的表达变化[J].法医学杂志.2015年,31(1):11-14.
[10]Yasuda Y,Tateishi N,Shimoda T,et al.Relationship between S100beta and GFAP expression in astrocytes during infarction and glial scar formation after mild transient ischemia[J].Brain Res,2004,1021(1):20-31.
[2]薛庆善,郭宛华.大鼠大脑皮质星形胶质细胞的体外培养及其神经突起生长作用研究[J].神经解剖学杂志,1996,12(2):151-155.
[3]鄂征.组织培养和分子细胞学技术[M].北京:北京出版社,1995:132.
[4]Mc Carthy KD,de Vellis J.Preparation of separate astroglial and oligo-dendroglial cell cultures from rat cerebral tissue[J].J Cell Biol,1980,85(3):890-902.
[5]薛庆善,肖渝平.神经胶质细胞的体外培养方法[J].解剖科学进展,1999,5(2):110-117.
[6]Rothermundt M,Peters M,Prehn JH,et al.S100B in brain damage and neurodegeneration[J].Microsc Res Tech,2003,60(6):614-632.
[7]Sofroniew MV,Vinters HV.Astrocytes:biology and pathology[J].Acta Neuropathol,2010,119(1):7-35.
[8]张彤,张祥建,刘瑞春,等.胶质纤维酸性蛋白与中枢神经系统疾病[J].脑与神经疾病杂志,2005,13(6):466.
[9]吴雨虹,王慧君,王欣.大鼠原发性脑干损伤后S100B和GFAP的表达变化[J].法医学杂志.2015年,31(1):11-14.
[10]Yasuda Y,Tateishi N,Shimoda T,et al.Relationship between S100beta and GFAP expression in astrocytes during infarction and glial scar formation after mild transient ischemia[J].Brain Res,2004,1021(1):20-31.