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目的研究米诺环素(Minocycline)对大鼠嗜铬细胞瘤细胞(Pheochromocytoma,PC12)氧糖剥夺损伤后的保护作用及血红素加氧酶-1(Heme Oxygenase-1,HO-1)表达的影响。方法体外培养高分化的PC12细胞,分别氧糖剥夺(Oxygen Glucose Deprivation,OGD)2,4,6,8 h筛选出稳定的细胞缺氧缺糖损伤模型。将细胞随机分为正常对照组(Con组),氧糖剥夺组(OGD组),OGD+米诺环素治疗组(MC组)及OGD+米诺环素+HO-1抑制剂锌原卟啉组(Znpp组),氧糖剥夺再复氧24 h后采用四甲基偶氮唑蓝(MTT)法检测PC12细胞存活率,RT-PCR和Western blotting法检测各组HO-1基因及蛋白的表达水平。结果 MTT法测得PC12细胞的光密度值随氧糖剥夺时间延长而逐渐降低,2 h时细胞存活率显著降低,6 h时更明显。米诺环素呈浓度依赖性显著提高氧糖剥夺后PC12细胞的存活率,其中1μM浓度最佳。Znpp能逆转米诺环素对PC12细胞氧糖剥夺损伤的保护作用。与对照组和氧糖剥夺组相比,米诺环素可显著上调HO-1 m RNA和蛋白表达。结论米诺环素对氧糖剥夺PC12细胞具有保护作用,其机制可能与上调HO-1表达有关。
Objective To investigate the protective effect of Minocycline on the oxidative stress-deprivation injury of rat pheochromocytoma (PC12) and the expression of heme oxygenase-1 (HO-1) influences. Methods Well-differentiated PC12 cells were cultured in vitro, and stable cell models of hypoxia-glucose deprivation injury were screened at 2,4,6,8 h after OGD (OGD). The cells were randomly divided into normal control group (Con group), OGD group (OGD group), OGD + minocycline group (MC group) and OGD + minocycline + HO-1 inhibitor zinc protoporphyrin group (Znpp group). After oxygen deprivation and reoxygenation for 24 h, the survival rate of PC12 cells was detected by MTT assay. The expression of HO-1 gene and protein in each group were detected by RT-PCR and Western blotting Level. Results The optical density of PC12 cells measured by MTT method decreased gradually with the prolongation of the time of OGD. The cell viability decreased significantly at 2 hours and became more obvious at 6 hours. Minocycline significantly increased the survival rate of PC12 cells after glucose deprivation in a concentration-dependent manner, with the best concentration being 1μM. Znpp can reverse the protective effect of minocycline on oxygen-glucose deprivation injury in PC12 cells. Minocycline significantly upregulated HO-1 mRNA and protein expression compared with control and OGD groups. Conclusion Minocycline has a protective effect on oxygen-glucose deprivation of PC12 cells, which may be related to the up-regulation of HO-1 expression.