沉默视黄酸核受体α损伤大鼠原代海马神经元的钙兴奋性

J4 ›› 2011, Vol. 37 ›› Issue (6): 976-980.

沉默视黄酸核受体α损伤大鼠原代海马神经元的钙兴奋性

江伟^1,2,3, 喻琴^1,2, 龚敏^1,2, 毕扬^1,2, 张赟^1,2, 陈立^1,2, 瞿平^1,2, 魏小平^1,2, 刘友学^1,2, 陈洁^1,2, 李廷玉^1,2

(1. 重庆医科大学附属儿童医院儿童营养中心|重庆400014；2.重庆医科大学儿童发育疾病省部共建教育部重点实验室|重庆400014；3.重庆医科大学附属儿童医院康复中心|重庆400014)

收稿日期:2011-06-29 出版日期:2011-11-28 发布日期:2011-11-28
通讯作者: 李廷玉 E-mail:（Tel: 023-63623604，E-mail: tyli@top.sina.com）
作者简介:江伟（1981-）|男|四川省达州市人|医学博士|医师|主要从事脑损伤及神经发育研究。
基金资助:
国家自然科学基金重点项目资助课题(30830106);国家自然科学基金青年基金资助课题(81100454)

Calcium excitability of rat primary hippocampal neuron damaged by silenced retinoic acid receptor α

JIANG Wei^1,2,3, YU Qin^1,2, GONG Min^1,2, BI Yang^1,2, ZHANG Zan^1,2, CHEN Li^1,2, QU Ping^1,2, WEI Xiao-Ping^1,2, LIU You-Xue^1,2, CHEN Ji^1,2, LI Ting-Yu^1,2

(1. Children Nutrition Center,Children&rsquo|s Hospital,Chongqing Medical University,Chongqing 400014,China;2. Key Laboratory of Developmental Diseases in Childhood,Ministry of Education,Chongqing Medical University,Chongqing 400014,China;3. Rehabilitation Center,Children’s Hospital,Chongqing Medical University,Chongqing 400014|China)

Received:2011-06-29 Online:2011-11-28 Published:2011-11-28

摘要/Abstract

摘要：

目的：了解视黄酸核受体α (RARα)对大鼠神经元功能的必要性。方法：采用组织消化原代贴壁法分离培养大鼠原代海马神经元，利用腺病毒载体特异沉默RARα；利用Real-Time PCR分析沉默RARα对神经元视黄酸(RA)信号各受体以及神经细胞标志物的影响；利用活细胞钙影像分析沉默RARα对神经元钙兴奋性的影响。结果：免疫荧光显示，分离培养的细胞90%表达神经元标志神经元特异性烯醇化酶(NSE)，腺病毒转染效率可达80%。PCR结果显示，RARα沉默后RARα表达降低75%(P<0.01)，其他受体均显著降低(P<0.01)，但RARβ显著上
调(P<0.05)。活细胞钙影像显示，沉默组钙兴奋性显著降低(P<0.05)，全反式视黄酸(ATRA)预处理24 h能显著增强钙兴奋性(P<0.01)。结论：RARα的缺失能显著降低原代海马神经元的神经元标志物NSE的表达，并显著损伤神经元的钙兴奋性。

关键词: 原代海马神经元；视黄酸；视黄酸核受体α；钙兴奋性；基因沉默

Abstract:

bstract:Objective
To study the necessary of retinoic acid receptor α (RARα) for rat neuron function.
Methods Tissue digestion was used to isolate and cultivate the rat primary hippocampal neurons,and the adenovirus vector was used to specifically silence the RARα;Real-Time PCR was used to analyze the influence of silenced RARα in retinoic acid(RA) receptors and the markers of nerve cells;live cell imaging analysis was performed to analyze the influence of the calcium excitability of neurons silenced RARα.Results The immunofluorescence results showed that 90% of the isolated cells expressed the neuron marker neuron-specific enolase (NSE),the adenoviral transfection efficiency was up to 80%.The PCR results showed the expression of RARα in silenced RARα neuron was decreased by 75% (P<0.01),the other receptors were significantly decreased (P<0.01),but RARβ was significantly increased (P<0.05).The live cell calcium imaging results showed the calcium excitability in silent group was significantly reduced (P<0.05),however all-trans retinoic acid (ATRA) pretreatment for 24 h could significantly enhance the calcium excitability (P<0.01).Conclusion The absence of RARα can significantly reduce the neuron marker NSE expression of the primary hippocampal neurons,and significantly damage the neuronal calcium excitability.

Key words: primary hippocampal neurons；retinoic acid；retinoic acid receptor α；calcium excitability；gene silence

中图分类号:

R338

江伟, 喻琴, 龚敏, 毕扬, 张赟, 陈立, 瞿平, 魏小平, 刘友学, 陈洁, 李廷玉. 沉默视黄酸核受体α损伤大鼠原代海马神经元的钙兴奋性[J]. J4, 2011, 37(6): 976-980.

JIANG Wei, YU Qin, GONG Min, BI Yang, ZHANG Zan, CHEN Li, QU Ping, WEI Xiao-Ping, LIU You-Xue, CHEN Ji, LI Ting-Yu. Calcium excitability of rat primary hippocampal neuron damaged by silenced retinoic acid receptor α[J]. J4, 2011, 37(6): 976-980.