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目的:研究α-酮戊二酸钠对CHO-hGPCRc细胞内钙离子浓度的影响,并分析其变化的原因,从功能上进一步鉴定人源G蛋白偶联受体hGPCR c表达细胞的可靠性。方法:将重组表达质粒pcDNA3.1(+)-hGPCRc转染入CHO-K1细胞,经G418(800μg/m l)作用7~10 d,挑选、扩增阳性克隆,得到CHO-hGPCR c细胞,再以RT-PCR检测所得细胞内hGPCRc mRNA的表达;然后用不同浓度的α-酮戊二酸钠诱导该细胞,通过激光扫描共聚焦显微镜观察F luo-3标记细胞内钙离子变化,以及细胞外钙离子对其影响。结果:(1)RT-PCR结果表明,所得CHO-hGPCR c细胞能够稳定表达hGPCR c的mRNA;(2)α-酮戊二酸钠可引起细胞内钙离子浓度的变化,表现为钙波动幅度明显增加,并呈浓度依赖性;(3)α-酮戊二酸钠引起的细胞内钙波动,不是细胞外钙离子内流所致,而是细胞内钙库动员的结果。结论:α-酮戊二酸钠为hGPCRc的特异性配基,CHO-hGPCRc细胞能够稳定表达具有功能活性的hGPCRc受体,为进一步开展hGPCRc研究奠定了基础。
OBJECTIVE: To study the effect of α-ketoglutarate sodium on the intracellular calcium concentration in CHO-hGPCRc cells and to analyze the reason for the change. The reliability of human G protein-coupled receptor hGPCR c cell line was further identified by its function. Methods: The recombinant plasmid pcDNA3.1 (+) - hGPCRc was transfected into CHO-K1 cells. The positive clones were selected and amplified by G418 (800μg / ml) for 7-10 days to obtain CHO-hGPCR c cells The expression of hGPCRc mRNA was detected by RT-PCR. The cells were induced by different concentrations of sodium α-ketoglutarate. The changes of F luo-3 labeled intracellular calcium were observed by laser scanning confocal microscopy. Effect of calcium ions on it. Results: (1) The results of RT-PCR showed that the obtained CHO-hGPCR c cells could stably express hGPCR c mRNA; (2) Sodium α-ketoglutarate induced changes of intracellular calcium concentration, Significantly increased and in a concentration-dependent manner; (3) Intracellular calcium mobilization induced by α-ketoglutarate was not caused by the influx of extracellular calcium ions but was the result of intracellular calcium mobilization. CONCLUSION: Sodium α-ketoglutarate is the specific ligand of hGPCRc. CHO-hGPCRc cells can stably express hGPCRc receptor with functional activity, which lays the foundation for further research on hGPCRc.