缺氧对体外培养的大鼠脑星形胶质细胞存活的影响

目的 研究缺氧对体外培养的大鼠星形胶质细胞活性及损伤的影响,确定星形胶质细胞体外培养的最佳缺氧时间点以对其进行深入研究。方法 体外培养大鼠大脑皮层星形胶质细胞,纤维酸性蛋白免疫荧光染色鉴定。通以5%CO₂+95%N₂混合气体造成缺氧,分别观察缺氧不同时间段星形胶质细胞光镜下的形态变化,死亡细胞数目,培养上清液的氧分压、葡萄糖和乳酸盐的浓度及乳酸脱氢酶(LDH)活性的变化。结果 星形胶质细胞在缺氧条件下,随着时间延长,细胞逐渐出现肿胀、漂浮和坏死。与对照组相比,缺氧10h组死亡细胞数、葡萄糖和乳酸盐的浓度及乳酸脱氢酶(LDH)活性的变化均有明显差异(P<0.05),且与缺氧时间呈正相关,其中缺氧组细胞培养上清液中葡萄糖浓度比对照组明显降低,而乳酸盐浓度和LDH活性则明显升高,而缺氧8h组虽然葡萄糖和乳酸盐的浓度及LDH活性的变化明显,但星形胶质细胞保存着多突触的形态。结论 缺氧可致体外培养的鼠脑星形胶质细胞损伤甚至死亡,缺氧8h可作为以体外培养的鼠脑星形胶质细胞为研究对象的缺氧生物学研究的最佳时间点。

Effect of hypoxia on the viability of rat brain astrocytes in vitro

Objective To investigate the effect of hypoxia on the viability of rat brain astrocytes in vitro and determine the optimal duration of hypoxic treatment for studying the biological behavior of the in vitro cultured cells in response to hypoxia. Methods Rat astrocytes were cultured and identified by glial fibrillary acidic protein (GFAP) immunofluorescent staining. After incubation with 5% CO2 95% N2 for different time to induce hypoxia, the cells were harvested and observed micros- copically for morphological changes and counting of the dead cells. The culture media of the cells were also collected and pO2, concentrations of glucose and lactate as well as lactate dehydrogenase (LDH) activity were analyzed. Results As the hypoxia prolonged, the astrocytes appeared swollen and floating in the medium with some becoming necrotic. Compared with the cells in the control group, the changes in the number of necrotic cells, concentrations of glucose and lactate, and LDH activity in the cells of the hypoxic group were significantly different after 10 h of hypoxia (P<0.05). However, the flat and polygonal morphology of the astrocytes almost remained unchanged after 8 h of hypoxia in spite of significant changes in the concentrations of glucose and lactate and LDH activity. Conclusion Hypoxia induced injury and necrosis of rat brain astrocytes in vitro, and 8 h of hypoxia can be the optimal time point for studying the biological behaviors of the cells in response to hypoxia.