活细胞核浆粘滞系数的荧光相关谱研究

Nucleoplasmic Viscosity of Living Cells Investigated by Fluorescence Correlation Spectroscopy

细胞核浆粘滞系数直接反映一定生理状态下核内微环境的特性,是判断病变细胞和研究核内分子功能机制的重要依据。为了实时、灵敏和无损地测定单个活细胞的核浆粘滞系数,提出了一种基于荧光相关谱(fluorescence correlation spectroscopy,FCS)技术的新型检测方法。研究中利用FCS技术测定绿荧光蛋白分子(EGFP)在细胞核内的扩散系数,并且根据Stokes-Einstein关系式计算出核浆粘滞系数,同时实现了对特定生理条件下核浆粘滞系数的跟踪测量。结果表明:在pH 7.4以及37℃的条件下,人肺腺癌细胞(ASTC-a-1)和人宫颈癌细胞(HeLa)的核浆粘滞系数分别是(2.55±0.61)cP和(2.04±0.49)cP,与传统方法的测量结果一致,并且发现核浆粘滞系数大于细胞质的粘滞系数。以上研究表明FCS技术可精确无损地检测单个活细胞内达飞升量级的微观环境,是探索细胞内生物分子动态行为的有力工具。

In order to non-invasively investigate nucleoplasmic viscosity in real time with good temporal resolution, the present study firstly introduced a new method based on fluorescence correlation spectroscopy （FCS）. FCS is a kind of single-molecule technique with high temporal and spatial resolution to analyze the dynamics of fluorescent molecules in nanomolar concentration. Through a time correlation analysis of spontaneous intensity fluctuations, this technique in conjunction with EGFP as a probe is capable of determining nucleoplasmic viscosity in terms of Stokes--Einstein equation as well as its corresponding analysis of the diffusion coefficient for EGFP in the nucleus. The results showed that nucleoplasmie viscosity of ASTC-a-1 cells and HeLa cells were respectively （2.55±0. 61） cP and （2.04±0. 49） cP at pH 7.4 and 37 ℃, consistent with the results by traditional meth- ods, and nucleoplasmic viscosity was found to be larger than cytoplasmic viscosity. Meanwhile, the real-time analysis of nucleoplasmic viscosity in living cells exposed to hypotonic media proved that FCS could be used to track the changing rheological characteristics of the nueleoplasm in living cells. Taken together, this study suggests that FCS provides an accurate and non-invasive method to investigate the mieroenvironment in living cells on the femtoliter scale and it can be used as a powerful tool in researches on the dynamical processes of intracellular molecules.