外源性Nurr1基因过表达增强SK-N-SH细胞对神经毒素6-OHDA敏感性的研究

The effects of overexpression of Nurr1 on vulnerability of SK-N-SH cells to neurotoxin 6-OHDA

目的 通过比较自身不表达Nurrl基因的人神经母细胞瘤细胞SK—N—SH和过表达外源性Null基因的SK—N—SH／Nurrl细胞对神经毒素6-羟多巴胺（6-OHDA）损伤的敏感程度，探讨Null基因与6一OHDA引起的多巴胺（DA）能神经元特异性损伤的相关性。方法①绘制SK—N—SH细胞及SK—N—SH／Nurrl细胞的生长曲线，比较外源性Null基因过表达对细胞增殖率的影响。②用不同浓度的6-OHDA（5—100μmol·L^-1）分别作用两株细胞1—24h，倒置相差显微镜下观察细胞形态变化，MTT测定法比较两株细胞的存活率。③用75μmol·L^-1的6-OHDA作用12h，经Hoechst33342染色，荧光显微镜观察细胞核形态，比较两株细胞的凋亡情况。结果①生长曲线结果显示，SK—N—SH／Null细胞增殖数目低于SK—N—SH细胞一②MTT结果显示，6-OHDA对两株细胞的存活均有抑制作用，但SK—N—SH／Null细胞存活率下降更为明显。③Hoechst33342染色结果显示，用75μmol·L^-1的6-OHDA作用12h后SK—N—SH细胞核没有出现明显的凋亡形态改变，而部分SK—N—SH／Null细胞的细胞核出现了染色质凝聚、碎裂等凋亡形态改变，比例约为22％-24％。结论外源性Null基因过表达抑制SK—N—SH细胞增殖，增强SK—N—SH细胞对6-OHDA的敏感性并有可能促进6-OHDA诱导的细胞凋亡。Null基因可能作为易感因子参与多巴胺能神经元的特异性损伤。

Aim To investigate whether Nurrl gene, a member of the nuclear receptor superfamily of transcription factors, contributed to 6-OHDA-induced DAergic neurons insults by comparing the effect of 6-OHDA on neuroblastoma cells SK-N-SH and SK-N-SH cells overexpressing Nurrlgene （ SK-N-SH/Nurrl ）. Methods The effect of Nurrl gene on cell proliferation of SK-N-SH cells was investigated, then SK-N-SH and SK-N-SH/Nurrl cells were exposed to 6-OHDA（5- 100μmol·L^-1） for 1 - 24 h, cells＇ morphology was observed under phase-contrast microscope. The cells viability was analyzed via MTT assay. Apoptosis was detected using Hoechst33342 staining. Results The Nurrl-overexpressing SK-N-SH cells showed significantly slow growth rate compared with that of SK-N-SH cells. Treatment with 50μmol · L^-1 6-OHDA changed SK-N-SH-Nurrl cells＇ morphology within 1 h, accompanied by retraction of processes, shrinkage of cell body, whereas the morphology of SK-N-SH cells changed after treated with 50μmol · L^-1 6-OHDA for 2 h. The result of MTT assay indicated that exposure to 100μmol · L^-1 6-OHDA for 24 h induced a significant decrease in SK-N-SH/Nurrl cells survival comparedwith SK-N-SH cells at various time points with an IC50 value of 75 μmol · L^-1 and 50μmol·L^-1 respectively in SK-N-SH and SK-N-SH/Nurrl cells. The evidence that treatment with 75μmol·L^-1 6-OHDA for 12 h induced typical apoptosis in SK-N-SH/Nurrl cells was found in morphological features provided by Hoechst33342 staining, including chromatin condensation and nuclear fragmentation, and the percentages of apoptosis in SK-N-SH/Nurrl cells was about 22% 24%. In contrast, no morphological characteristics of apoptosis in SK-N-SH cells were observed. Conclusions The present study suggests that Nurrl gene renders SK-N-SH cells more vulnerable to neurotoxic insuits. The mechanism of such action is probanly that normal Nurrl gene function can stimulate apoptotic pathway induced by 6-OHDA, and play a major role in the high sensitivity of DArgic neurons to 6-OHDA insuits as a vulnerable factor.