亚低温对星形胶质细胞缺氧后缝隙连接功能的影响

Effects of Mild Hypothermia on Gap Junctional in Astrocyte under Hypoxia

目的观察亚低温对缺氧条件下星形胶质细胞缝隙连接变化的影响,探讨亚低温对脑缺血的保护机制。方法原代培养星形胶质细胞,传至第3代以神经胶质纤维酸性蛋白(GFAP)免疫细胞化学染色鉴定星形胶质细胞,采用三气培养箱分别调节温度,缺氧12 h。分为正常对照组(对照组)、常温(37.0 oC)缺氧组、亚低温(32.0 oC)缺氧组。用锥虫蓝染色法测定各组缺氧后星形胶质细胞存活率;应用划痕标记染料示踪技术在激光扫描共聚焦显微镜下测定缺氧12 h荧光黄染料在缝隙连接间的扩散距离;细胞免疫荧光技术检验缺氧星形胶质细胞缝隙连接的主要缝隙连接蛋白成分Cx43表达水平。结果常温缺氧组部分细胞边缘皱缩;亚低温缺氧组经亚低温干预后大部分细胞轮廓较清晰,胞周光晕明显,细胞存活力较常温缺氧组增加;对照组、常温缺氧组及亚低温缺氧组死亡细胞数分别为(6.19±0.29)个、(8.54±0.99)个和(6.89±2.41)个,对照组死亡细胞数最少,组间两两比较均差异有统计学意义(P<0.05);常温缺氧组荧光黄染料仍可在细胞间进行扩散,扩散距离和Cx43荧光强度较对照组无明显变化(P>0.05);亚低温缺氧组与对照组和常温缺氧组比较,荧光染料扩散距离减少、Cx43荧光强度降低,差异有统计学意义(P<0.05)。结论亚低温可以减轻体外培养星形胶质细胞的缺氧损伤;降低Cx43表达而抑制缝隙连接功能,可能是亚低温干预的脑保护机制之一。

ABATRACT Aim To observe the changes of astrocyte gap junction and influence mild hypothermia under the condition of hypoxia. Methods The primary astrocytes were isolated from the cortex of SD fetal rats（less than 24 h） and identified by immunocytochemical analysis of anti-glia fibrillary acid protein（GFAP） after 3 passages. Astrocytes of passage 3 were divided into a control group, a normothermic group and a mild hypothermia group in a controlled atmosphere culture chamber for 12 h, which was sealed to maintain the gas composition and kept the temperature at 37.0 oC or 32.0 oC. Trypan blue staining was used to detect the rate of survived astrocytes by inverted phase contrast microscope as cellular damaged index. The effects of mild hypothermia on gap junctional intercellular communication（GJIC） in astrocyte were determined of lucifer yellow scrape-loading and dye transfer（SLDT） technique by laser scanning confocal microscope（LSCM）. Immunocytochemistry was used to examine the expression of connexin 43（Cx43） in the astrocytes of every group after hypoxia. Results The morphology of culture astrocytes creased on edge in normothermic group after hypoxia, however, the body of mild hypothermia group was little change. With regard to the number of death astrocytes, the survival rate of control group was the highest in the three groups, there was significant difference between the normal group and mild hypothermia group after 12 h exposure to hypoxia conditions（the control group： 6.19±0.29, normothermic group： 8.54±0.99, mild hypothermia group： 6.89±2.41, P〈0.05）, After 12 h of hypoxia in 37.0 oC, the fluorescence distance of GJIC and the expression of Cx43 were showed, and there was no difference between the control group and normothermic group（P〈0.05）, which indicated a tendency to decrease in mild hypothermia group compared to the control and normothermic group（P〈0.05, P〈0.01）. Conclusion Mild hypothermia can relieve the deterioration of astrocytes by hypoxia. Mild hypothermia may be one of the brain protection mechanisms by reducing Cx43 expression and inhibiting the gap junction.