论文部分内容阅读
目的研究多巴胺(DA)对原代星形胶质细胞谷氨酸(Glu)摄取能力的影响,以及DA通过痕量胺相关受体1-兴奋性氨基酸转运体2(TAAR1-EAAT2)信号通路对星形胶质细胞Glu摄取能力的影响。方法采用Amplex Red谷氨酸测定试剂盒测定经过DA处理的原代星形胶质细胞对Glu摄取含量的变化,反转录PCR检测TAAR1、EAAT2 mRNA水平,Western blot法检测TAAR1、EAAT2蛋白水平;采用TAAR1小干扰RNA(siRNA)和TAAR1质粒转染DA处理后的原代星形胶质细胞,Western blot法检测EAAT2表达水平并采用Amplex Red谷氨酸测定试剂盒测定培养上清液Glu含量。结果 DA处理的原代星形胶质细胞EAAT2水平降低,TAAR1水平增加,培养上清液Glu含量上升。DA处理经TAAR1 siRNA转染后的原代星形胶质细胞,上调EAAT2水平,培养上清液中Glu含量减少;DA处理经TAAR1质粒转染后的原代星形胶质细胞,则EAAT2降低,培养上清液中Glu的含量增加。结论 DA通过影响星形胶质细胞TAAR1-EAAT2信号通路,减弱细胞Glu摄取能力,引起细胞外Glu蓄积,从而损伤星形胶质细胞的功能。
Objective To investigate the effect of dopamine (DA) on the uptake of glutamate (Glu) in primary astrocytes and the effect of DA on TAAR1-EAAT2 signaling pathway Effect of Astrocyte Glu Uptake. Methods Amplex Red glutamate assay kit was used to determine the change of Glu uptake by primary astrocytes treated with DA. The mRNA levels of TAAR1 and EAAT2 were detected by reverse transcriptase PCR and the levels of TAAR1 and EAAT2 by Western blot. TAAR1 small interfering RNA (siRNA) and TAAR1 plasmid transfected DA-treated primary astrocytes, Western blot was used to detect EAAT2 expression level and Amplex Red glutamate assay kit determination of the culture supernatant Glu content. Results The level of EAAT2 in primary astrocytes decreased, the level of TAAR1 increased and the content of Glu in culture supernatant increased. DA treatment of primary astrocytes transfected with TAAR1 siRNA upregulated the level of EAAT2, and the content of Glu in the culture supernatant decreased. In DA treated with TAAR1 plasmid, the level of EAAT2 was decreased in primary astrocytes , The culture supernatant Glu content increased. Conclusion DA can impair the function of astrocytes by affecting the ability of Glu uptake of astrocytes by affecting the TAAR1-EAAT2 signaling pathway, leading to accumulation of extracellular Glu.