低氧反应元件介导的低氧调控的神经营养因子-3表达上调减少低氧诱导的PC12细胞凋亡  

Up-regulated expression of neurotrophin-3 mediated by hypoxia response element attenuates apoptosis induced by hypoxia in PC12 cells

作　　者：张军峰[1] 史利利[1] 张力[1] 赵朝华[1] 杨蓬勃[2] 张建水[2] 刘勇 徐曦[1] 

机构地区：[1]西安医学院人体解剖学教研室,西安710021 [2]西安交通大学医学部神经生物学研究所,西安710061

出　　处：《解剖学报》2015年第5期581-586,共6页Acta Anatomica Sinica

基　　金：国家自然科学基金资助项目(81301041);陕西省教育厅专项科研计划资助项目(2013JK0756;14JK1614);陕西省青年科技新星资助项目(2015KJXX-43);陕西省优势学科建设经费资助项目[陕(2014)3号-1001];西安医学院博士基金资助项目(2012DOC02)

摘　　要：目的在体外培养的PC12细胞中观察低氧反应元件(HRE)介导的神经营养因子-3(NT-3)对低氧反应性表达上调及其对低氧诱导的PC12细胞凋亡的影响。方法用分子生物学方法将5拷贝HRE(5HRE)和NT-3克隆入反转录病毒载体中构建HRE介导的低氧调控表达载体,并转导入PC12细胞,ELISA法检测NT-3的表达和分泌,TUNEL法检测细胞凋亡,Western blotting检测p38丝裂原活化蛋白激酶(p38MAPK)和Caspase-3激活情况。结果成功构建重组反转录病毒载体,并将外源基因转导入PC12细胞中(PC12-NT3-EGFP、PC12-5HRE-NT3-EGFP和PC12-5HRE-EGFP)。在正常条件培养下,PC12-5HRE-NT3-EGFP细胞中NT-3表达水平较低,但在低氧处理后,NT-3表达明显升高(n=3,P<0.05)。在低氧处理后,与PC12细胞组相比,PC12-5HRE-NT3-EGFP组中细胞凋亡明显减少(n=3,P<0.05),且p38 MAPK和Caspase-3磷酸化也明显减少(n=3,P<0.05)。结论在PC12细胞中HRE介导的低氧反应性调控NT-3的表达上调可以对PC12细胞产生保护作用。Objective To investigate the controlled expression of neurotrophin-3 （NT-3） by HRE under hypoxic conditions and determine the protective effects of conditionally expressed NT-3 on hypoxia-induced apoptosis in PC12 cells. Methods Five copies of the HRE （5HRE） and NT-3 were employed to construct a therapeutic vector, and transferred into PC12 ceils. Expression and secretion of NT-3 were detected by ELISA. Apoptosis of PC12 cells induced by hypoxia was assayed by TUNEL. Activation of p-38 and Caspase-3 was detected by Western blotting. Results The retroviral vectors were successfully constructed and transfected into PC12 cells to produce gene transferred cells, PC12-NT3-EGFP, PCt2-SHRE-NT3-EGFP and PC12-SHRE-EGFP. Compared with normal conditions, in which NT-3 was expressed at low levels, the expression of NT-3 significantly increased under hypoxic conditions in PC12-5HRE-NT3-EGFP （n = 3, P 〈 0. 05）. The conditional adjustment of NT-3 expression by 5HRE significantly reduced apoptosis induced by hypoxia in PC12-SHRE-NT3-EGFP （ n = 3, P 〈 O. 05）. In addition, the hypoxia-induced phosphorylation of both p38 and Caspase-3 activities was decreased in PC12-SHRE-NT3-EGFP under hypoxic conditions （ n = 3, P 〈 O. 05 ）. Conclusion Up- regulated expression of NT-3 mediated by hypoxia response element in response to hypoxia in PC12 cells can protect PC12 ceils against hypoxia-induced apoptosis.

关 键 词：低氧反应元件 神经营养因子-3 低氧 免疫印迹法 

分 类 号：R338[医药卫生—人体生理学]