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

作　　者：刘扬[1] 赵咏梅[2] 张海燕[3] 李卫红[3] 岳云[1] 

机构地区：[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

出　　处：《首都医科大学学报》2011年第4期525-533,共9页Journal of Capital Medical University

基　　金：国家自然科学基金项目(30270433)~~

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(50～100μmol/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75μmol/L 6-OHDA作用12 h,通过透射电镜(transmission electronmicroscopy,TEM)观察细胞超微结构变化。用Western blotting方法检测75μmol/L6-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,75μmol/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100μmol/L6-OHDA作用6、12 h后,2株细胞均表现为毒性损伤。TEM显示,75μmol/L6-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。4.75μmol/L6-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(≤75μmol/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。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.

关 键 词：核相关受体因子 过表达 6-羟基多巴胺 凋亡 流式细胞术 

分 类 号：R332.8[医药卫生—人体生理学]