星形胶质细胞体外缺血缺氧损伤后炎症模型的建立及应用

目的 建立SD大鼠星形胶质细胞缺血缺氧损伤后炎症模型,探讨亚低温对星形胶质细胞缺血缺氧及损伤后炎症反应的影响.方法 体外原代培养新生SD大鼠星形胶质细胞,免疫荧光染色检测胶质纤维酸性蛋白(GFAP)进行鉴定.实验分正常对照组(C组)、单纯缺氧组(H组)和缺氧+白细胞组(H+W组),以无糖DMEM培养基、5%CO2+95%N2混合气体培养4 h诱导细胞缺氧模型.H+W组加入1 mLSD大鼠外周血白细胞(1×106/mL),C组加入等量培养液.将3组细胞分别置入37℃、34℃、32℃、30 ℃ CO2孵箱中作用24 h,应用速率法和LIVE/DEAD染色分别检测3组细胞在不同温度下乳酸脱氢酶(LDH)释放率的变化和形态学变化.结果 缺氧4 h即可造成星形胶质细胞的损伤.37℃时C组、H组、H+W组细胞LDH释放率依次升高,差异有统计学意义(P<0.05);与37℃相比,亚低温状态(34℃、32℃)下H组、H+W组细胞LDH释放率均降低;但在30℃时,则有明显升高,与32℃相比差异具统计学意义(P<0.05).结论 亚低温状态可明显降低星形胶质细胞的缺血缺氧性损伤及炎症性损伤,其机制可能并非通过单纯的抑制代谢来实现的.

Establishment and application of an in vitro neruoinflammation model in astrocytes after ischemic injury

Objective To establish a in vitro cell model of inflammation following ischemic injury in rat astrocytes, and study the effects of mild hypothermia on the response of astrocytes to ischemia and inflammation. Methods Primary cultured neonatal SD rat astrocytes identified by GFAP immunofluorescence staining were divided into normal control group (C), hypoxia group (H), and hypoxia and leukocyte treatment group (H+W). Hypoxic injury of the cells was induced by exposure to glucose-flee DMEM medium in 5% CO2 and 95% N2 for 4 h, and for leukocyte treatment, the astrocytes were co-cultured with leukocytes isolated from rat blood. The 3 groups of cells were subjected to hypothermia treatments at 37, 34, 32 and 30 ℃, and the cell viability was estimated by lactic dehydrogenase (LDH) release assay and live/dead assay. Results Ischemic injury occurred in the astrocytes after 4 h of hypoxic exposure. Hypoxic treatment of the cells at 37 ℃ resulted in increased LDH release in all the 3 groups, and the increment intensified in the order of groups C, H and H+W. Mild hypothermia at 34 ℃ and 32 ℃ significantly reduced the LDH release in groups H and H+W (P<0.05), but hypothermia at 30 ℃ significantly increased LDH release in comparison with that at 32 ℃ (P<0.05). Conclusion Mild hypothermia can significantly reduce the severity of ischemic and inflammatory injuries in rat astrocytes possibly mediated also by other mechanisms in addition to inhibited astrocyte metabolism due to low temperatures.