摘要
ER-α36是一种分子量为36kDa的雌激素受体(estrogen receptor,ER)新亚型,其主要通过介导膜雌激素信号通路参与多种细胞生理、病理等过程。本文利用RNA干扰技术敲低大鼠肾上腺嗜铬细胞瘤细胞株PC12的ER-α36基因,研究ER-α36与Akt在神经细胞缺糖损伤中的关系。采用MTT、Western blot和Annexin V/PI双染等方法,观察了ER-α36在细胞缺糖应激反应中的作用。结果显示:(1)随着缺糖损伤时间的增加,PC12细胞的存活率逐渐降低,6h后其细胞存活率与对照组相比具有极显著性差异(P<0.01),凋亡率升高,6h后具有极显著性差异(P<0.01);葡萄糖转运蛋白Glut-4表达水平显著降低(P<0.01);(2)缺糖损伤3h时,ER-α36明显减少(P<0.01),但随后逐渐增加;缺糖可引起抗凋亡蛋白Akt磷酸化水平先升高后降低(P<0.05);加入PI3K抑制剂LY294002,ER-α36表达降低,Akt的激活被抑制;(3)敲低ER-α36后缺糖处理,细胞凋亡率高于野生型,具有极显著性差异(P<0.01);敲低ER-α36的细胞缺糖导致的Akt激活减弱,Caspase-3表达增多。以上结果表明,在PC12细胞的缺糖应激反应中,Akt激活情况可能与ER-α36相关,两者共同参与缺糖损伤应激调控。
ER-α36 is a novel 36-kDa variant of ER-α. A large of evidence demonstrated that ER-α36 responded to membrane-initiated estrogen signaling pathways which were involved in the physiological and pathological process in many kinds of cells. In this study, knock-down of ER-α36 expression in pheochromocytoma （PC12） cells （named as PC12-36L cells） by using the shRNA method was used to evaluate the relationship between ER-α36 and Akt in neurons under glucose deprivation. The effect of ER-α36 on outgrowth of PC 12 cells, as well as the neuroprotective effect of ER-α36 on injured PC 12 cells exposed to glucose deprivation was observed by us- ing MTT assay, Western blot and Annexin V/PI staining et al. The results showed that, （1） Glucose deprivation induced by MEM treat- ment for 6 h reduced survival rate and increased apoptotic rate in PC12 cells significantly compared to control group （P 〈 0.01）; and it produced a decrease in the expression of Glut-4 protein （P 〈 0.01）; （2） The expression level of ER-α36 was decreased significantly at 3 h of glucose deprivation, and then increased, while phosphorylation of Akt participated in the glucose deprivation was increased at first and then reduced; LY294002 （PI3K inhibitor） contributed to decreased expression of ER-α36, and suppressed the activation of Akt; （3） The rate of apoptosis was significantly increased in PC12-36L cells after glucose deprivation compared with that in wild type PC12 cells （P 〈 0.01）. Furthermore, phosphorylation of Akt was decreased and Caspase-3 was increased by glucose deprivation in PC12-36L cells com- pared with those in wild type PC12 cells. The study reveals that phosphorylation of Akt is associated with ER-α36 in PC12 cells exposed to glucose deprivation, and both are involved in the regulation of stress responses.
出处
《生理学报》
CAS
CSCD
北大核心
2013年第4期381-388,共8页
Acta Physiologica Sinica
基金
supported by the National Natural Science Foundation of China(No.30970353)
