海藻糖对生物人工肝用人永生化肝细胞系C3A细胞低温的保存

Trehalose in C3A hepatocytes hypothermic preservation

背景:随着生物人工肝在临床上的应用,需要有一种方便、有效、实用的保护生物反应器装载细胞活力及功能的方法,设计有自主知识产权的生物人工肝用肝细胞的低温保护液迫在眉梢。目的:验证海藻糖作为低温保护剂在4℃低温保存肝细胞的作用。方法:采用C3A人永生化肝细胞系在不同低温保存液(DMEM培养液,乳酸钠林格氏液,加有不同浓度海藻糖的乳酸钠林格氏液,UW液)4℃保存24,48,72h。复苏后行细胞活力、细胞损伤指标、氧自由基代谢相关指标等检测,并通过荧光双染法观察细胞凋亡情况。结果与结论:不同低温保存液保存不同时间C3A细胞的活力、细胞损伤及细胞凋亡表现均有所不同,其中同时间点不同浓度海藻糖组均优于单用乳酸钠林格氏液组,但不如UW液组(P<0.01)。而保存24h的不同浓度海藻糖组及UW液组与乳酸钠林格氏液组相比差异无显著性意义(P>0.05),提示海藻糖能有效减轻低温对肝细胞凋亡及缺血再灌注损伤的程度,可成为低温保存液中有效及重要的保护剂组分。

BACKGROUND：With the application of bioartificial liver in clinic,a convenient,effective,practical protection of bioreactor loaded cell viability and function method,and the design of proprietary bioartificial liver cells with hypothermic protective solution are urgently needed.OBJECTIVE：To verify the effect of trehalose as cryoprotectant in 4 ℃ C3A hepatocytes hypothermic preservation.METHODS：C3A hepatocytes were preserved in different hypothermic preservation solution for 24,48 and 72 hours at 4 ℃.The following preservation solutions were tested：1） Dullbecco＇s modified Eagle＇s medium（DMEM）,2） Lactated Ringer＇s solution,3） Lactated Ringer＇s solution with different concentration of trehalose,4） University of Wisconsin（UW） solution.The viability of hypothermic preserved C3A hepatocytes,index of cellular damage,oxygen-derived free radicals metabolic capacity of hypothermic preserved C3A hepatocytes after warm-reperfusion,and the apoptosis was observed by dual fluorescent staining method.RESULTS AND CONCLUSION：The viability of hypothermic preserved C3A hepatocytes,index of cellular damage,and apoptosis were different in different groups.The indexs in Lactated Ringer＇s solution with different concentration of trehalose were significantly better than Lactated Ringer＇s solution,but inferior to UW solution（P 0.01）.Such parameters in Lactated Ringer＇s solution with different concentration of trehalose and UW solution were no significant difference with Lactated Ringer＇s solution（P 0.05）.Trehalose in C3A hepatocytes hypothermic preservation can effectively lessen hepatocytes apoptosis and depressed the extent of ischemia-reperfusion injury,thus,trehalose is the effective and important component in hypothermic preservation solution.