黄芪提取物对冷冻保存原代人胎肝细胞的保护作用

The Protective Effect of Astragalus membranaceus Extraction on Cryopreserved Primary-cultured Human Fetal Hepatocytes

目的:观察黄芪提取物(AE)对原代人胎肝细胞经冻存复苏后活性变化的影响,以探索改善人肝细胞冻存效果的新方法。方法:采用二步灌注法分离人胎肝细胞,细胞分别加入由不同浓度AE配制的五种冻存保护剂(Ⅰ:10%DMSO;Ⅱ:5%DMSO+2 mg/L AE;Ⅲ:5%DMSO+20 mg/L AE;Ⅳ:5%DMSO+60 mg/L AE;Ⅴ:5%DMSO+100 mg/L AE),冷冻保存1个月。1个月后快速复苏,进行细胞活率、形态学及基本功能检测。结果:人胎肝细胞在经不同保护剂冻存复苏后,其活率、形态学及功能表现有所不同,其中Ⅳ、Ⅴ组复苏后细胞的存活率和24 h贴壁率,与Ⅰ组无显著差异(P>0.05),但显著高于Ⅱ、Ⅲ处理组(P<0.05);Ⅳ组复苏后细胞的白蛋白与天门冬氨酸转氨酶浓度测定、氯化铵转化实验结果显著优于其它各组(P<0.05)。结论:AE可对原代人胎肝细胞冷冻保存有保护作用,其中60 mg/L是最佳浓度;AE与二甲基亚砜联合使用降低了冻存保护剂DMSO的浓度,表明两者有协同作用。

Objective： To observe the influence of Astragalus membranaceus Extraction （AE） on the primary-cultured human fetal hepatocytes stored in liquid nitrogen and explore a new method for the cryopreservation of human hepatocytes with improved function. Methods ： Human fetal hepatocytes were harvested by two-step collagenase perfusion, and then stored in a liquid nitrogen for one month with five different cryoprotectants （ Ⅰ - 10% DMSO, Ⅱ.5 % DMSO ＋ 2mg/L AE,Ⅲ ：5% DMSO ＋ 20mg/L AE, Ⅳ ：5% DMSO ＋ 60mg/L AE, Ⅴ-5% DMSO ＋ 100mg/L AE）. One month later, the cells were thawed rapidly and the viability, morphology and basic function of them were tested. Results ： The human fetal hepatocytes in different groups showed various levels of viability, morphological manifes- tation and cell function respectively. After thawing, the viability rate and flash adhering rate in group Ⅳ and V had no significant difference with group Ⅰ （ P 〉 0. 05 ）, but were higher than group Ⅱ and Ⅲ（ P 〈 0. 05 ） ; the cell function analysis in the group Ⅳ, the results of ALB and AST level determination, NH4Cl transformation test, were the best among the groups（ P 〈 0. 05 ）. Conclusion ： AE can provide protection for human fetal hepatocytes in cryopreservation, and the best performance concentration level of its is 60mg/L;the preservation dosage of DMSO can be reduced when combined with AE in the preservation solution, which shows that AE has a synergistic effect with DMSO.