6-OHDA诱导过表达nurr1基因的SK-N-SH细胞凋亡

doi:10.3969/j.issn.1006-7795.2011.04.017

首都医科大学学报 ›› 2011, Vol. 32 ›› Issue (4): 525-533.doi: 10.3969/j.issn.1006-7795.2011.04.017

6-OHDA诱导过表达nurr1基因的SK-N-SH细胞凋亡

刘扬¹, 赵咏梅², 张海燕³, 李卫红³, 岳云¹

1. 首都医科大学北京朝阳医院麻醉科,北京 100020;2. 首都医科大学宣武医院中心实验室,教育部神经变性病重点实验室, 北京 100053;3. 首都医科大学细胞生物学教研室,北京 100069

收稿日期:2011-02-28 修回日期:1900-01-01 出版日期:2011-08-21 发布日期:2011-08-21

Effects of overexpression of nurr1 on apoptosis of SK-N-SH cells induced by neurotoxin 6-OHDA

LIU Yang¹, ZHAO Yong-mei², ZHANG Hai-yan³, LI Wei-hong³, YUE Yun¹

1. Department of Anesthesia, Beijing Chaoyang Hospital, Capital Medical University, Beijing 100020, China;2. Central Laboratory, Xuanwu Hospital, Capital Medical University, Key Laboratory of Neurodegenerative Diseases, Ministry of Education, Beijing 100053, China;3. Department of Cell Biology, Capital Medical University, Beijing 100069, China

Received:2011-02-28 Revised:1900-01-01 Online:2011-08-21 Published:2011-08-21

摘要/Abstract

摘要： 目的比较自身不表达核相关受体因子(nuclear receptor related factor1,Nurr1)基因的人神经母细胞瘤细胞株SK-N-SH和过表达外源性nurr1基因的SK-N-SH/Nurr1细胞对6-羟基多巴胺(6-hydroxydopamine,6-OHDA)的损伤反应,探讨nurr1基因在6-OHDA致细胞损害(死亡或凋亡)中的作用。方法用免疫细胞化学方法和RT-PCR检测基因转染细胞SK-N-SH/NURR1的nurr1蛋白及NURR1 mRNA的表达。细胞经不同浓度6-OHDA(50~100 μmol/L)作用不同时间(6、12 h),经AnnexinV/PI双染后流式细胞术(flow cytometry,FCM)测定早期细胞凋亡比例。细胞经75 μmol/L 6-OHDA作用12 h,通过透射电镜(transmission electron microscopy,TEM)观察细胞超微结构变化。用Western blotting方法检测75 μmol/L 6-OHDA作用6、12 h,细胞凋亡因子Caspase-3活性前体蛋白的表达水平。结果免疫细胞化学染色结果显示,基因转染细胞SK-N-SH/Nurr1表达NURR1蛋白;经RT-PCR检测,基因转染细胞表达NURR1 mRNA,说明外源性nurr1基因在转染细胞内过表达。FCM检测结果显示,75 μmol/L 6-OHDA作用12 h, SK-N-SH/Nurr1细胞凋亡比例明显高于SK-N-SH细胞(P=0.000), 而100 μmol/L 6-OHDA作用6、12 h后,2株细胞均表现为毒性损伤。TEM显示,75 μmol/L 6-OHDA作用12 h后,部分SK-N-SH/Nurr1细胞出现了典型凋亡改变,而SK-N-SH细胞主要表现为毒性损伤。4.75 μmol/L 6-OHDA作用6、12 h,SK-N-SH/Nurr1细胞Caspase-3活性前体蛋白表达显著下降(P=0.000),而SK-N-SH细胞Caspase-3活性前体蛋白表达变化不明显。结论外源性nurr1基因过表达促进6-OHDA诱导的SK-N-SH/Nurr1细胞凋亡,在低剂量(≤75 μmol/L) 时,凋亡比例与剂量呈正相关,高剂量时6-OHDA主要诱导细胞毒性损伤。

Abstract: Objective To investigate the possible correlation between the expression of Nurr1 gene, a midbrain transcription factor, and selective apoptotic cell death induced by neurotoxin 6-OHDA. Methods Firstly, the expression of Nurr1 was detected by immunohistochemistry and RT-PCR in transfected SK-N-SH/Nurr1 cells. Secondly, following treatment with 50~100 μmol/L 6-OHDA on both SK-N-SH and SK-N-SH/Nurr1 cells for 6 and 12 h, the cell loss was detected to discriminate between apoptosis and necrosis by using flow cytometry(FCM) and Annexin V/PI double staining. Thirdly, the ultrastructural changes of both cells were observed by transmission electronic microscopy(TEM) after treatment with 75 μmol/L 6-OHDA for 12 h. Finally, the level of apoptosis effector Caspase-3 of both cells were detected after exposure to 75 μmol/L 6-OHDA for 6 and 12 h by Western blotting analysis. Results Firstly, immunohistochemical staining and RT-PCR analysis showed that the over expression of Nurr1 in transfeced SK-N-SH cells. Secondly, Annexin V/PI double staining with FCM showed treatment with 75 μmol/L 6-OHDA for 12 h induced a significant apoptotic phase with Annexin V+/PI-only in SK-N-SH/Nurr1 cells(P=0.000), while both cells showed increased necrotic phase with Annexin V+/PI+ after exposure to 100 μmol/L 6-OHDA for 6 and 12 h. Thirdly, the result of TEM showed that treatment with 75 μmol/L 6-OHDA for 12 h induced typical apoptosis in SK-N-SH/Nurr1 cells. In contrast, no morphological characteristics of apoptosis in SK-N-SH cells were observed. Finally, Western blot analysis showed that treatment with 75 μmol/L 6-OHDA for 6 and 12 h decreased the level of pro-Caspase-3 in SK-N-SH/Nurrl cells but not in SK-N-SH cells(P=0.000). Conclusion Nurr1-overexpression stimulated apoptotic pathway induced by 6-OHDA,specifically in a dose-dependence manner.

Key words: nuclear receptor related factor1, overexpression, 6-hydroxydopamine, apoptosis, flow cytometry

中图分类号:

R 332-8

刘扬;赵咏梅;张海燕;李卫红;岳云. 6-OHDA诱导过表达nurr1基因的SK-N-SH细胞凋亡[J]. 首都医科大学学报, 2011, 32(4): 525-533.

LIU Yang;ZHAO Yong-mei;ZHANG Hai-yan;LI Wei-hong;YUE Yun. Effects of overexpression of nurr1 on apoptosis of SK-N-SH cells induced by neurotoxin 6-OHDA[J]. Journal of Capital Medical University, 2011, 32(4): 525-533.

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6-OHDA诱导过表达nurr1基因的SK-N-SH细胞凋亡

Effects of overexpression of nurr1 on apoptosis of SK-N-SH cells induced by neurotoxin 6-OHDA

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