白介素-1β对MN9D细胞Nurr1及酪氨酸羟化酶表达的影响

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。

Effect of interleukin-1β on Nurr1 and tyrosine hydroxylase expression in MN9D cells

The objectives of this study are to investigate whether interleukin-1 beta(IL-1β)could increase Nurr1 expression in a dopamine-synthesizing cell line(MN9D) with immature characteristics and to study whether tyrosine hydroxylase(TH) expression is up-regulated in response to endogenous Nurr1-overexpression,in order to investigate the role of Nurr1 during the development of dopaninergic neurons.The MN9D cells were treated with 20 ng/ml IL-1β for 2 h,4 h and 6 h.The changes of MN9D cells'morphology were observed under phase-contrast microscope.The expressions of Nurr1 and TH in MN9D cells after IL-1β treatment were analyzed by immunohistochemical staining and Western blot.The results showed that the morphology of MN9D cells did not change significantly after IL-1β treatment for 2,4 and 6 h compared with that of MN9D cells left untreated.Nurr1-positive staining of MN9D cells treated with IL-1β for 2,4 and 6 h was much stronger than that of untreated cells,while the percent of TH-positive stained MN9D cells was similar to that of untreated(P>0.05).Western blot result showed that after treated with IL-1β for 2 h,4 h and 6 h,the expression of Nurr1 protein in MN9D cells was 233%,232% and 254% of untreated MN9D cells(P<0.05),while the expression of TH protein was 97%,107% and 105% of untreated MN9D cells(P>0.05).The present results suggest that IL-1β up-regulates Nurr1 protein expression in MN9D cells within 2-6 h but does not increase TH expression.Up-regulation of Nurr1 expression alone might be insufficient for activating TH expression in MN9D cells,but rather requires specific cellular(or neuronal) environments or cofactors.