对肝细胞及肝病变细胞进行荧光光谱特性研究, 能为早期筛查与攻克肝癌提供光谱学依据。 实验目的包括, 通过光谱实验获得细胞特异性荧光光谱； 结合流式细胞仪获得最大饱和荧光强度与细胞直径的相关性。 实验过程, 首先使用荧光光谱仪来检测肝细胞、 肝纤维细胞以及两种肝癌细胞； 采用去拉曼散射的方法消除背景噪声, 获得五种浓度下细胞荧光光谱； 其次, 使用流式细胞仪检测四种细胞直径的大小, 根据双参数散点图分析四种细胞的直径特点； 最后, 利用高斯多峰拟合对比光谱差异, 并且分析细胞直径对荧光饱和强度变化趋势的影响, 得出细胞大小对荧光饱和强度非线性拟合曲线的影响规律。 光谱实验发现, 在550~750 nm之间, 肝细胞存在两个特异性荧光峰, 第一个峰值位于592 nm处, 第二个峰位于682 nm处, 且前者明显高于后者； 肝癌, 肝纤维细胞除具备与肝细胞相同位置的两个峰外, 在726 nm处出现第三个特异性荧光峰, 并在592 nm处获得最大激发光强, 在726 nm处的荧光峰高于在682 nm处的第二个荧光峰； 结合高斯多峰拟合法对峰值和各峰位置, 以及峰型展宽进行分析。 肝癌细胞和肝纤维细胞三个峰的展宽基本相同, 正常肝细胞最大激发峰展宽略小于另三种细胞, 但是682 nm处的小峰展宽略大于病变细胞； 流式细胞仪实验结果显示, 肝癌细胞直径大于肝纤维细胞大于肝细胞, 同种浓度下荧光强度也是肝癌细胞高于肝纤维细胞高于肝细胞； 利用非线性曲线拟合细胞最大荧光强度随浓度变化曲线, 根据曲线斜率的变化规律, 发现四种细胞的最大荧光强度会随着细胞浓度增大而增强, 但是逐渐呈现荧光饱和状态。 随着细胞直径增加, 最大荧光强度饱和趋势更为明显, 单个细胞自激发效率降低。 结果显示, 将细胞形态学与光谱学有机的融合, 结合两种分析方式, 能提高细胞判断的准确性和有效性。 通过对肝细胞、 肝癌细胞以及肝纤维细胞的荧光光谱特性进行研究, 并结合细胞直径分析荧光饱和变化趋势, 能够为肝病变细胞的研究提供一定的光谱学依据。

Researched the fluorescence spectral characteristics of hepatic cell, hepatoma carcinoma cell and hepatic fibrosis cell to provide spectroscopy basis for early screening of liver cancer. The purpose of the experiment included the detection of cells by fluorescence spectrometer to acquire specific fluorescence spectra; eliminate background noise by de-Raman scattering to acquire fluorescence spectra of five cell concentrations； the cell diameter was detected by flow cytometry, analysis of the diameter characteristics of four cells based on two-parameter scatterplot; combined with Gauss multi-peak fitting parameter analysis results. The experimental process began as hepatic cell, hepatic fibrosis cell and two hepatoma carcinoma cell were detected by fluorescence spectroscopy and analyzed by flow cytometry as well. Then, Gauss multi-peak was used to fit the fluorescence spectrum differences, and the difference among the cells was analyzed. Finally, the fluorescence saturation intensity nonlinear fitting curve was compared to analyze the effect of cell size on it. The results showed that there were two specific fluorescence peaks in the hepatic cell between 550 and 750 nm. Combined with Gaussian multi-peak fitting, the peak height, peak center and peak width were analyzed. The results showed, the first peak was at 592 nm and the second peak was at 682 nm, and the former was significantly higher than the latter. In hepatoma carcinoma cell and hepatic fibrosis cell there was a third specific fluorescence peak at 726 nm except for two peaks at the same position as hepatic cell, and the maximum excitation intensity was obtained at 592 nm, and the fluorescence peak at 726 nm was higher than the second peak at 682 nm. The width of hepatoma carcinoma cell and hepatic fibrosis cell were basically the same. The maximum excitation peak width of hepatic cell was slightly smaller than that of the other two cells, but the small peak width at 682 nm was slightly larger than that of the diseased cells. The results of flow cytometry showed that the diameter of hepatoma carcinoma cell was the largest diameter, and hepatic fibrosis cell was larger than hepatic cell. Through fitting the curve of the fluorescence saturation intensity trend of the cell with the concentration curve by the nonlinear curve and analyzing the slope of the curve, the results showed that the fluorescence saturation intensity trend of the four cells increased with the increase of the cell concentration, but gradually showed the fluorescence saturation state. As the cell diameter increased, the trend of maximum fluorescence saturation intensity was more obvious, and auto fluorescence spectrum efficiency of single cells decreased. The results showed that the rational combination of cell morphology and spectroscopy, combined with two methods of analysis, improved the accuracy and effectiveness of cell judgment. By studying the fluorescence spectrum characteristics of hepatic cell, hepatoma carcinoma cell, hepatic fibrosis cell, and analyzing the fluorescence saturation intensity in combination with cell diameter, it can provide a certain spectral basis for the study of liver disease cells.