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目的探讨环巴胺(Cyclopamine)对恶性胶质瘤细胞株LN229帕金森相关基因Nurr1表达的影响。方法在脂多糖(LPS)刺激LN229细胞的条件下,运用实时定量PCR检测SHH通路基因PTCH、Gli1及Nurr1基因mRNA含量,Western Blotting检测LPS对LN229细胞Nurr1蛋白表达的影响。进一步在Cyclopamine阻断Sonic Hedgehog(SHH)信号通路的条件下,实时定量PCR及Western Blotting检测Nurr1的mRNA含量及其蛋白的表达及下游炎症因子IL-1βmRNA的含量。结果 LPS刺激下LN229细胞SHH通路相关基因mRNA,及Nurr1 mRNA升高,Western Blot结果进一步证明LPS可以促进Nurr1蛋白表达。Cyclopamine阻断组与对照组比较,在转录水平和翻译水平明显促进了Nurr1基因表达升高,及下游炎症因子IL-1βmRNA的含量。结论 Cyclopamine对LN229细胞Nurr1表达的影响可能与帕金森发病过程中胶质细胞的活化有关。
Objective To investigate the effect of cyclopamine on the expression of Parkinson’s gene Nurr1 in human glioma cell line LN229. Methods The mRNA expressions of PTCH, Gli1 and Nurr1 in SHH pathway were detected by real-time quantitative PCR under the condition of lipopolysaccharide (LPS) stimulation of LN229 cells. The effect of LPS on the expression of Nurr1 protein in LN229 cells was detected by Western Blotting. The mRNA and protein expression of Nurr1 and IL-1βmRNA were also detected by real-time quantitative PCR and Western Blotting after Cyclopamine blocked the Sonic Hedgehog (SHH) signaling pathway. Results LPS-induced SHH pathway-related gene mRNA and Nurr1 mRNA increased in LN229 cells. Western Blot results further proved that LPS can promote the expression of Nurr1 protein. Compared with the control group, Cyclopamine blockade group significantly increased the expression of Nurr1 gene and the content of IL-1βmRNA in the transcriptional level and translation level. Conclusion The effect of Cyclopamine on the expression of Nurr1 in LN229 cells may be related to the activation of glial cells in the process of Parkinson’s disease.