人宫颈癌细胞(Hela)内的^(31)P核磁共振研究

Study on Intracellular of Hela Cells by ^(31)P Nuclear Magnetic Resonance

建立了无损伤性31PNMR研究细胞内物质的实验方法,并对人宫颈癌细胞(Hela)的31PNMR谱中含磷小分子代谢物的谱峰进行了分析;细胞内无机磷(Pi)的化学位移对pH非常敏感,通过测定其化学位移可间接确定细胞内的pH,Hela细胞内Pi峰的化学位移为5.88±0.01(n=3),计算得到细胞内pH值为7.05±0.01;通过测量Hela细胞的31PNMR谱中ATP的α磷和β磷及γ磷的化学位移差值,得出Hela细胞内Mg2+与ATP结合的复合物MgATP和整个ATP量的比值,计算得到Hela细胞内游离Mg2+浓度为(253.3±0.13)mmol/L(n=3),与其它分析方法相比,31PNMR测定细胞内游离Mg2+浓度具有对细胞样品无损伤的优点.

A ^31p nuclear magnetic resonance （NMR） method has been established to noninvasively measure intracellular condition of Hela cell, stable ^31p NMR spectra of living cells were obtained and intracellular metabolite in Hela of phosphorized small molecule cell was analyzed. The chemical shift of intracellular inorganic phosphate （Pi） ,which is very sensitive to cytosolic pH, can be shifted on the ^31p NMR spectrum as pH is changed and cytosolic pH can be derived from chemical shift of Pi peak. Intracellular pH 7.05±0.01 was calculated based on the chemical shift of Pi peak （5.88±0.01） in Hela cells. Based upon a measurement of chemical shift differences between the α and β-phosphoryl resonances in the ^31p NMR spectrum of intracellular adenosine triphosphate （ATP） in Hela cell, the fraction of total ATP complexed to Mg^2＋ was estimated, while cytosolic free magnesium ion concentration in Hela cells was measured nondestructively to be 253.3 mmol/L. ^31p nuclear magnetic resonance method has noninvasive virtue in measuring intracellular Mg^2＋ compared with other technique.