摘要目的 建立一种稳定的成人肝细胞冷冻方法,为构建永生化肝细胞系提供充足的肝细胞来源,为肝细胞移植、生物人工肝支持系统治疗急、慢性肝病及肝细胞体外应用模型提供潜在的肝细胞资源.方法 20例供肝采用离体两步胶原酶灌注技术分离成人原代肝细胞.选择7个预置时间点(2、6、12、24、36、48、72 h),在4℃人无血清培养基预置12 ～ 24 h后,使用海藻酸钠-多聚赖氨酸微球微囊化肝细胞,液氮保存.分析预置微囊冷冻组及对照组肝细胞的形态学、病理结构、白蛋白水平、尿素合成、细胞周期、肝细胞特异性功能基因mRNA和蛋白表达水平.各组间数据比较均采用方差分析,计数资料采用Fisher 's exact test检验.结果 在部分肝叶切除后使用离体两步胶原酶灌流技术分离所得肝细胞活率和贴壁率分别是75.0％±4.6％和72.0％±6.0％.4℃预置12h或24h细胞的白蛋白分泌高于其他时间点,F=40.3,P值均＜0.05,差异有统计学意义.4℃预置12h或24h与立即冷冻(IC)组比较,解冻后贴壁肝细胞白蛋白和尿素合成水平明显增高,且肝细胞特异性功能基因mRNA和蛋白水平明显增高,此两个时间点是最适预置时间.预置微囊冷冻(PMC)组细胞与IC组比较,肝细胞形态保持的更为完整,细胞增殖周期、糖原染色及肝细胞功能特异性基因的mRNA和蛋白表达水平均明显增强.PMC组细胞在贴壁培养后第2、3、4天的白蛋白分泌分别与直接培养(DC)组分别比较,P值均＞ 0.05,差异均无统计学意义;而PMC组细胞在贴壁培养后第2、3、4天,与IC组的白蛋白水平比较(119.2 ng/ml对比101.2 ng/ml,110.0 ng/ml对比87.6 ng/ml,98.2 ng/ml对比73.8 ng/ml)和尿素合成比较(7.83μg/ml对比6.79 μ g/ml,6.83 μ g/ml对比5.89μg/ml,5.85 μg/ml对比4.83 μg/ml)均较IC组明显增高,两组比较,F值分别为22.1、31.1,P值均＜0.05,差异均有统计学意义.结论 部分肝叶切除后使用离体两步胶原酶灌注技术提供了一种创新、简单、可靠的肝细胞分离技术,人肝细胞在微囊化冷冻前4℃预置12 ～ 24h可更好地恢复形态和功能的完整性,为药理毒理学、生物人工肝以及细胞治疗的临床应用提供可能.

abstractsObjective To establish a stable method of isolation,culture and cryopreservation of adult primary hepatocytes to provide potential hepatocyte resources for therapeutic usage in hepatocyte transplantation and bioartificial liver support systems for the treatment of acute and chronic liver diseases,and for experimental usage as an in vitro model of the liver.Methods Adult hepatocytes from 20 human donors undergoing partial hepatectomy were isolated using a two-step extracoporeal collagenase perfusion technique.Seven preincubation time points (2h,6h,12h,24h,36h,48h and 72h) were selected for optimization.After pre-incubation at 4 ℃ for 12-24h in HepatoZYME-SFM (the optimal condition),hepatocytes were microencapsulated using alginate-poly-L-lysine-alginate microcapsules,transferred to a complete medium containing 10％ dimethyl sulphoxide and immediately placed into an isopropanol progressive freezing container for overnight freezing at -80 ℃ followed by immersion in liquid nitrogen the next day.During the post-thawing culture period,the cells were tested for albumin secretion,urea synthesis,cell cycling,transcription and protein synthesis (measuring mRNA and protein levels),and the morphological structure and pathology,for comparison with the features from before microencapsulated cryopreservation (PMC).Results The viability and plating efficiency of the hepatocytes isolated using the two-step extracorporeal collagenase perfusion technique were 75.0±4.6％ and 72.0±6.0％,respectively.The pre-incubation times of 12h and 24h (viability:61.4±4.8％ and 62.0±5.6％; plating efficiency:3.2±5.8％ and 62.6±3.6％,respectively) showed significantly higher albumin secretion than all other time points tested (F =40.3,all P ＜ 0.05).Compared with the immediate cryopreservation (immediately frozen control) hepatocytes,the PMC hepatocytes showed significantly better transcription and protein synthesis and higher albumin secretion and urea levels.The PMC group did not show a significantly different level of albumin production from the directly cultured hepatocytes (culture day 2:ll9.2ng/ml vs.131.36ng/ml,P =0.051; day 3:110ng/ml vs.120.4ng/ml,P=0.063; day 4:98.2ng/ml vs.109.8ng/ml,P ＞ 0.05).However,over culturing days 2,3 and 4,comparison of the PMC hepatocytes to the immediate cryopreservation hepatoeytes showed the former to have significantly higher secretion of albumin (119.2ng/ml vs.101.2ng/ml,110.0ng/ml vs.87.6ng/ml and 98.2ng/ml vs.73.8ng/ml; all P ＜ 0.05) and urea level (7.83 μg/ml vs.6.79 μg/ml,6.83 μg/ml vs.5.89 μg/ml and 5.85 μg/ml vs.4.83 μg/ml; all P ＜ 0.05).The post-thawed PMC hepatoeytes showed preservation of the morphological structure,while the immediate cryopreservation hepatocytes did not.Conclusion The two-step extracorporeal collagenase perfusion technique after partial hepatectomy is a novel,simple,and reliable method for hepatocyte isolation.Pre-incubation at 4 ℃ for 12-24h before the microencapsulation cryopreservation allows for efficient recovery of functional and morphological integrity after thawing and provides viable hepatoeytes that may be useful for clinical applications in pharmacotoxicology,bioartificial liver therapy and cell therapy in humans.