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利用具有未成熟特性的多巴胺(DA)合成细胞系MN9D,观察白介素-1beta(IL-1β)对MN9D细胞内源性孤儿核受体Nurr1表达的作用,并研究Nurr1表达增高对MN9D细胞酪氨酸羟化酶(TH)表达的影响,探讨Nurr1调控DA能神经元发育的机制。用20 ng/ml的IL-1β作用于MN9D细胞,于IL-1β作用的2、4、6 h用相差显微镜观察细胞形态的变化,并采用免疫细胞化学染色及Western blot方法检测IL-1β作用后MN9D细胞内源性Nurr1蛋白表达的变化,以及Nurr1表达变化对MN9D细胞TH表达的影响。结果显示:经20 ng/ml的IL-1β作用2、4、6 h,MN9D细胞的形态与未经IL-1β作用的细胞相比没有明显改变。从加入IL-1β2 h起,直至6 h,MN9D细胞Nurr1免疫荧光染色强度比未加IL-1β作用的正常对照组细胞明显增强,但此时MN9D细胞中TH阳性细胞的百分比与正常对照组相比没有明显改变(P>0.05)。Western blot结果显示:20 ng/ml的IL-1β作用2、4、6 h,MN9D细胞Nurr1蛋白表达分别为233%、232%和254%,经统计学检验,与正常对照组相比具有显著性差异(P<0.05)。而IL-1β作用2、4、6 h,MN9D细胞TH蛋白的表达分别为97%、107%和105%,但经统计学检验,与正常对照组相比没有显著性差异(P>0.05)。本研究结果表明,IL-1β在2～6 h可迅速明显激活MN9D细胞内源性Nurr1的表达,但此时单纯Nurr1表达增加并不影响MN9D细胞TH的表达;TH的表达激活可能还需要除Nurr1外的其它特殊的细胞(或神经元)的环境或因子的共同作用。
The effect of interleukin-1beta (IL-1beta) on the expression of Nurr1 in orphaned nuclear receptor of MN9D cells was observed by using MN9D cell line with immature characteristic dopamine (DA), and the effect of Nurr1 expression on MN9D cell tyrosine Hydroxylase (TH) expression of Nurr1 to explore the mechanism of DA neurons development. The morphological changes of MN9D cells were induced by 20 ng / ml IL-1β at 2, 4 and 6 h, and the effect of IL-1β was detected by immunocytochemistry and Western blot The change of endogenous Nurr1 protein expression in MN9D cells and the effect of Nurr1 expression on the TH expression in MN9D cells. The results showed that the morphology of MN9D cells did not change significantly when treated with 20 ng / ml IL-1β for 2, 4, and 6 h compared with those without IL-1β. The intensity of Nurr1 immunofluorescence in MN9D cells was significantly increased from 2 h after adding IL-1β to 6 h, but the percentage of TH-positive cells in MN9D cells was significantly higher than that in normal control group Than no significant change (P> 0.05). The results of Western blot showed that Nurr1 protein expression in MN9D cells was 20%, 232% and 254%, respectively, at 20 ng / ml IL-1β for 2, 4 and 6 h, which was significantly higher than that of the normal control Sex differences (P <0.05). However, the expression of TH protein in MN9D cells was 97%, 107% and 105% respectively after 2, 4 and 6 h of IL-1β. However, there was no significant difference between the two groups (P> 0.05) . The results of this study showed that IL-1β could rapidly activate the endogenous Nurr1 expression in MN9D cells at 2 ~ 6 h, but the increase of Nurr1 alone did not affect the TH expression in MN9D cells; the activation of TH might also need to be removed Nurr1 other special cells (or neurons) of the environment or factors together.