应用胎鼠皮质神经元建立缺血再灌注损伤模型

摘要

ObjectiveBy using primary cultured neuronal to develop an in vitro model of simulated ischemia-reperfusion(IR) injury.MethodsImmunofluorescence was used to verify neuronal. Neuronal was iminersed in mineral oil and after 30 min replace the whole culture medium,collecting cells and extracting RAN at different time after medium replacement.The expression of cytokine was detected by using RT-PCR,ELISA was used to detection the expression of cytokine protein in supernatants were collected at 24 h after medium replacement,neuronal apoptosis was determined by flow cytometry.ResultsNeuronal was verified by MAP2 In neuronal of IR group after medium replacement the mRNA expression of cytokine was higher than in control group.In the IR group at 24 h after medium replacement the protein expression of cytokine in the supernatants was significantly higher than in the control group,at 6h, the apoptosis was obviously increased.ConclusionHigh purity of neuronal was achieved .The in vitro model of simulated ischmia-reperfusion(IR)injury in neuronal can be successfully created using paraffin oil.%目的：用原代培养的胎鼠皮质神经元建立缺血再灌注（IR）模型。方法取孕18~21d的c57BL/6胎鼠大脑皮层，用胰酶消化后进行培养，用免疫荧光法进行鉴定。神经元用石蜡油覆盖，一定时间后更换全培养基，之后在不同时间点收集细胞提取RNA，用RT-PCR法检测白细胞介素6（IL-6）、白细胞介素β（IL-1β）、肿瘤坏死因子α（TNF-α）mRNA的表达。结果神经元纯度>90%，与对照组比较，神经细胞在更换培养基后各细胞因子转录水平增加，更换培养基后24h上清液液中各细胞因子蛋白表达均明显增高，6h后，凋亡显著增加。结论可获得高纯度的原代神经元；用石蜡油可成功建立神经元体外模拟缺血再灌注模型。