解剖学报

解剖学报 ›› 2015, Vol. 46 ›› Issue (5): 581-586.doi: 10.16098/j.issn.0529-1356.2015.05.001

• 神经生物学 •    下一篇

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

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2 徐曦1*   

  1. 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
  • 收稿日期:2015-03-10 修回日期:2015-04-30 出版日期:2015-10-06 发布日期:2015-10-06
  • 通讯作者: 徐曦 E-mail:zhangjf_08@126.com
  • 基金资助:

    国家自然科学基金;陕西省教育厅专项科研计划项目;陕西省教育厅专项科研计划项目

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

ZHANG Jun-feng1 SHI Li-li1 ZHANG Li1 ZHAO Zhao-hua1 YANG Peng-bo2 ZHANG Jian-shui2 LIU Yong2 XU Xi 1*   

  1. 1. Department of Anatomy, Xi’an Medical University, Xi’an 710021, China; 2. Institute of Neurobiology, Xi’an Jiaotong University Health Science Center, Xi’an 710061,China
  • Received:2015-03-10 Revised:2015-04-30 Online:2015-10-06 Published:2015-10-06
  • Contact: XU Xi E-mail:zhangjf_08@126.com

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。

关键词: 低氧反应元件, 神经营养因子-3, 低氧, 免疫印迹法

Abstract:

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 cells. 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, PC12-5HRE-NT3-EGFP and PC12-5HRE-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 PC12-5HRE-NT3-EGFP (n=3, P<0.05). In addition, the hypoxiainduced phosphorylation of both p38 and Caspase-3 activities was decreased in PC12-5HRE-NT3-EGFP under hypoxic conditions (n=3, P<0.05). Conclusion Up-regulated expression of NT-3 mediated by hypoxia response element in response to hypoxia in PC12 cells can protect PC12 cells against hypoxia-induced apoptosis.

Key words: Hypoxia response element, Neurotrophin-3, Hypoxia, Western blotting