摘要
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 examine the effects of a-ketoglutarate sodium on the intracellular calcium concentration of CHO-hGPCRc, a cell line expressing hGPCRc, and further confirm the reliability of CHO-hGPCRc used for hGPCRc functional exploration. Methods： Cell line CHO-K1 was transfected with pcDNA3. 1 （ ＋ ）-hGPCRc and then selected with G418 at 800 μg/ml for 10 - 14 days. After transfection and selection, the cell line CHO-hGPCRc was identified by RT- PCR for hGPCRc mRNA stable expression. Stimulated by α-ketoglutarate sodium at a concentration ranging from 0 to 100 μmol/ml, CHO-hGPCRc cells were detected by laser scanning confoeal microscopy for their [ Ca^2＋ ]i ichanges. Meanwhile, the influence of extracellular Ca^2＋ addition on[ Ca^2＋ ]i Of CHO-hGPCRc cells was also scanned. Results： The cell line CHO-hGPCBc could stably express hGPCBc mRNA indicated by BT-PCR detection. When stimulated with different concentrations Of α-ketoglutarate sodium, CHO-hGPCRc cells showed marked increase in [ Ca^2＋ ]i in a concentration dependent manner. Pre-chelation of extracellular Ca^2＋ using EGTA could not influence the above effects of a-ketoglutarate sodium on [Ca^2＋ ]i-Conclusion： As the ligand of hGPCBc, α-ketoglutarate sodium could enhance [Ca^2＋ ]i of CHO-hGPCBc not by promoting extracellular calcium entrance but by mobilizing intracellular calcium storage. Further exploration on signal tranaduction mediated by hGPCBc can be carried out with the cell line CHO-hGPCBc.
出处
《军事医学科学院院刊》
CSCD
北大核心
2007年第1期39-41,共3页
Bulletin of the Academy of Military Medical Sciences
