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目的:建立犬肾细胞(Madin-Darby canine kidney,MDCK)细胞缺氧模型,进一步阐明甘氨酸对缺氧细胞增殖活性的影响及其作用机制。方法:将MDCK细胞置于体积分数为95%N2和5%CO2的有机玻璃调节性密闭容器中,分别培养24、36、48、72或84 h,用四甲基偶氮唑盐(MTT)法检测甘氨酸对缺氧性损伤MDCK细胞增殖活性的影响。将MDCK细胞分为正常组、缺氧组和甘氨酸处理组,加药后孵育1、2或3 h后,收集细胞总蛋白,用Western blot检测细胞外信号调节激酶(extracellular signal-regulated kinase 1 and 2,ERK1/2)、p38MAPK和Akt的磷酸化活性。结果:在所观察的所有缺氧时段内,MDCK细胞MTT活性均较正常对照组明显下降(P<0.01)。加入甘氨酸后,缺氧24、36或48 h后细胞的增殖能力比缺氧组有明显增强,差异有统计学意义。在缺氧72或84 h后,甘氨酸未能显示明显的保护作用。缺氧时ERK1/2和Akt的磷酸化活性明显降低,p38MAPK的磷酸化活性明显增高。将甘氨酸加入到缺氧细胞中,ERK1/2和Akt又重新被激活,p38MAPK被抑制。结论:甘氨酸可保护MDCK细胞免于早期缺氧性损伤,该作用可能通过激活ERK1/2和Akt,抑制p38MAPK而实现。
OBJECTIVE: To establish an animal model of hypoxia induced by Madin-Darby canine kidney (MDCK) cells and to further clarify the effect of glycine on hypoxic cell proliferation and its mechanism. Methods: MDCK cells were placed in a glass container with a volume of 95% N2 and 5% CO2 for 24, 36, 48, 72 or 84 h, respectively. The cells were treated with MTT, Assay was used to examine the effect of glycine on the proliferation of hypoxic injury MDCK cells. The MDCK cells were divided into normal group, hypoxia group and glycine group. After incubated for 1, 2 or 3 h, the total cellular protein was collected and the expression of extracellular signal-regulated kinase 1 and 2, ERK1 / 2), phosphorylation of p38MAPK and Akt. RESULTS: The MTT activity of MDCK cells was significantly lower than that of the normal control during all hypoxic periods observed (P <0.01). After addition of glycine, the proliferation ability of cells after hypoxia for 24, 36 or 48 h was significantly higher than that of hypoxia group, the difference was statistically significant. Glycine failed to show significant protective effect after 72 or 84 h of hypoxia. Hypoxia ERK1 / 2 and Akt phosphorylation activity was significantly reduced p38MAPK phosphorylation activity was significantly higher. Glycine was added to hypoxic cells, ERK1 / 2 and Akt were reactivated, p38MAPK was inhibited. CONCLUSION: Glycine can protect MDCK cells from early hypoxia injury. This effect may be through activating ERK1 / 2 and Akt and inhibiting p38MAPK.