大鼠皮层神经元和星形胶质细胞原代培养、鉴定及氧糖剥夺模型的制备
Primary culture identification of rat cortical neurons and astrocytes and establishment of oxygen glucose deprivation model
目的：进行大鼠皮层神经元和星形胶质细胞的原代培养和鉴定，并建立大鼠皮层神经细胞OGD模型。方法：进行大鼠神经元和星形胶质细胞原代培养后，分别采用NSE、GFAP免疫细胞化学染色对两种细胞进行鉴定，将两种细胞进行缺氧处理后用台盼蓝染色及LDH漏出率检测衡量细胞损伤程度。结果：神经元培养至第6d、星形胶质细胞培养第9d，NSE、GFAP染色阳性细胞分别为86.32%±2.64%、98.54%±2.01%，神经元OGD15min、30min，台盼蓝染色所有神经细胞均拒染，90min后神经元胞浆空泡明显，蓝染细胞明显增多，180min OGD使神经元全部蓝染，星形胶质细胞 OGD 15min~180min均未能对细胞形态和活性产生明显影响；随OGD时间增加，神经元和星形胶质细胞 LDH漏出率逐渐增加，OGD 60min时，神经元和星形胶质细胞LDH漏出率开始明显增加（p<0.05）。结论：成功完成大鼠皮质神经元和星形胶质细胞原代培养，神经元和星形胶质细胞分别在培养第6天和第9天可用于模型制备，成功建立神经元和星形胶质细胞氧糖剥夺模型。
基础医学细胞生物学神经病学、精神病学
原代培养氧糖剥夺神经元星形胶质细胞
徐忠信,莽靖,李昕华,邢影,邵延坤,何金婷.大鼠皮层神经元和星形胶质细胞原代培养、鉴定及氧糖剥夺模型的制备[EB/OL].(2010-01-11)[2025-10-31].http://www.paper.edu.cn/releasepaper/content/201001-342.点此复制
Objective: To culture and identify the rats’ cerebral cortical astrocytes and neurons, and to set up the OGD model of cortical neurocytes. Methods: Cell presented in the culture were shown to be astrocytes and neurons after characterization by immunocytochemistry, using a primary specific anti-GFAP, NSE antibody. Cell culrures were subjected to OGD injury using hypoxic D-Hank’s, and were incubated in an anaerobic chamber which was refitted from a vacuum dryer. Cortial cultures were deprived of oxygen and glucose for 15, 30, 45, 60, 90, 120 and 180min. The rate of cell death and LDH release were detected. Results: Neurons exposed to 45-60min OGD showed some cells blue-stained. Many neurons body were swollen after 90min OGD. By exposed to 180min OGD, 100% neurons were stained by trypan blue. The astrocytes were not stained during the period of exposure to OGD from 15 to 180min. The LDH release was dependent on the duration of OGD exposure. 30min OGD caused the release of LDH from cultures of neurons and astrocytes, and 60min OGD caused a significant increase on the levels of LDH release (p<0.05). Conclusions: The culture of rats’ cerebral cortical astrocytes and neurons were accomplished by combined enzymatic digestion and mechanical dissociation. Neurons and astrocytes could be explored to OGD at the 6th and 9th day, respectively. The model of OGD ischemia in neurocytes were established by hypoxic D-Hank’s and an anaerobic chamber.
primary cultureOGDneuronsantrocytes
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