用流式细胞仪测定人肝细胞系DNA指数值作为其生物功能筛选指标的研究

A research on the DI by FCM as an screening measurement of the human liver cell lines' biological function

目的 了解DNA指数 (DI)与人肝细胞系生物功能之间的关系。探讨DI值可否作为人工肝生物材料的初步筛选指标。方法 收集国内 9株人工肝细胞系 ,分别编号为细胞系 1- 9(CL1-9)。分别培养后获取细胞。取正常人全血分离淋巴细胞 ,作为正常二倍体对照。以流式细胞仪(FCM)测定 9株人肝细胞系DI值作为分化指标 ,判断人肝细胞系的分化情况。人肝细胞系分别接种1.2× 10 6个细胞 ,收集其培养生长旺盛、生物功能良好的第 5天时的培养上清液 ,测定上清液中的尿素、人白蛋白和安定含量 ,计算细胞系尿素合成、人白蛋白合成和安定转化量。进行DI值和肝功能指标间的相关性分析。CX7自动生化分析仪测定尿素浓度 ;用人白蛋白放免试剂盒经改良后采用放射免疫抗原竞争法测定人白蛋白浓度 (人白蛋白与牛血清白蛋白无交叉反应 ) ;培养上清中另加入 4 μg/ml安定作肝细胞药物代谢功能指标 ,提取后以 75 2型紫外分光光度计测定安定含量。采用双变量相关与回归分析方法进行统计学分析。结果 人肝细胞系DI值与人肝细胞系尿素合成量、人白蛋白合成量及安定转化量均呈线性相关 ,相关系数分别为 - 0 .94 37、- 0 .8795、- 0 .9386。结论 CL1分化程度最高 ,生物功能最好 ;

Objective To investigate the relationship between DNA Index (DI) and the human liver cell lines' biological function and discuss whether DI can be used as a screening parameter of the human liver cell lines' biological function. Methods 9 human liver cell lines were collected, and named separately from 1 to 9 (Cell Line 1 to Cell Line 9, abbreviated as CL1 to CL9). Culture the cells and collect them separately. The lymophocytes separated from the normal human blood are used as a control of the normal diploid. To judge the differentiation degree of the human cell lines, their DI indexes are determined by flow cytometry (FCM). 1.2×10 6 cells of each cell line were separately inoculted in the plates respectively. As a probe of the metabolism function of the liver cells, 4 μg/ml diazepam was added in the culturing system. The supernant was collected respectively on day 5 when the cells were proliferating abundantly, and the biological function were determined. The concentrations of urea, human albumin and diazepam in the supernant were determined of the cells and the amount of the urea, human albumin and diazepam synthesized or transformed by the cells were calculated, the correlation between the DI and the liver function was investigated. Double variances correlation and regression analysis was adopted as statistical method. Results There were linear correlation between the DIs of human liver cell lines and their biological function. Correlation coefficient was -0.9437, -0.8795, -0.9386 respectively. Conclusions The cell line of CL1, whose differentiation degree is the best, have the supreme biological function. DI may be used as a preliminary index in chosing the biomaterials of BLA from human liver cell lines.