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 史利利 张力 赵朝华 杨蓬勃 张建水 刘勇 徐曦*

doi:10.16098/j.issn.0529-1356.2015.05.001

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

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*

作者信息 +

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*

Author information +

文章历史 +

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。

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.

导出引用

张军峰史利利张力赵朝华杨蓬勃张建水刘勇徐曦*. 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[J]. 解剖学报. 2015, 46(5): 581-586 https://doi.org/10.16098/j.issn.0529-1356.2015.05.001

ZHANG Jun-feng SHI Li-li ZHANG Li ZHAO Zhao-hua YANG Peng-bo ZHANG Jian-shui LIU Yong XU Xi*. Up-regulated expression of neurotrophin-3 mediated by hypoxia response element attenuates apoptosis induced by hypoxia in PC12 cells[J]. Acta Anatomica Sinica. 2015, 46(5): 581-586 https://doi.org/10.16098/j.issn.0529-1356.2015.05.001

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