乳猪肝细胞的低温保存  

Cryopreservation of suckling pig hep- atocytes

作　　者：陈钟[1] 丁义涛[1] 张鹤云[2] 

机构地区：[1]南京大学医学院附属鼓楼医院肝胆外科,江苏省南京市210008 [2]南京大学生化系,江苏省南京市210093

出　　处：《世界华人消化杂志》2002年第2期173-176,共4页World Chinese Journal of Digestology

基　　金：江苏省卫生厅重点发展项目基金资助课题;No.BQ200020~~

摘　　要：目的:探讨简单有效的猪肝细胞低温保存方法.方法:采用改良原位两步胶原酶灌注法分离乳猪肝细胞.将肝细胞接种到含100mL·L-1二甲亚砜?激素?生长因子和新生小牛血清的RPMI1640培养基中,分别用直接冻存法(A组)和短期培养后冻存法(B组)保存10d.肝细胞复苏后在无血清培养基中培养5d,观察LDH漏出量?肝细胞活率?蛋白质?尿素合成功能?葡萄糖-6-磷酸酶活性及安定转化功能.同时测定新分离?培养肝细胞的上述指标作为对照组(C组).结果:两冻存组复苏后的肝细胞的活率(A组96.1%±19.4%,B组95.8%±20.0%vsC组98.9%±10.3%,P<0.05)?蛋白质合成功能(A组36.7±2.2μBq·cell-1,B组34.8±1.9μBq·cell-1vsC组38.5±2.7μBq·cell-1,P<0.05)?安定转化功能低于对照组(安定浓度:A组7.42±0.34mg·L-1,B组7.53±0.32mg·L-1vsC组6.47±0.20mg·L-1,P<0.05).两冻存组间LDH漏出量?肝细胞活率?蛋白质合成功能?安定转化功能及葡萄糖-6-磷酸酶活性无明显区别,A组尿素合成功能(3.36±0.29nmol·cell-1)低于B组(3.70±0.12nmol·cell-1).结论:将猪肝细胞接种到含100mL·L-1二甲亚砜?激素?生长因子和新生牛血清的RPMI1640培养基中,采用计算机程控的直接冻存和快速解冻方法,复苏后的肝细胞在无血清培养基中培养5d,其活率和功能除尿素合成功能较低外,与短期培养后冻存的肝细胞相仿.AIM:To explore a better and simpler cryopreservation method o f porcine hepatocytes. METHODS:Suckling pig hepatocytes were isolated by modified two- step in situ collagenase perfusion method. Hepatocytes were suspended in RPMI164 0 medium supplemented with 100mL·L-1 DMSO, hormone, growth factors and n ewborn bovine serum and cryopreserved for 10d with immediate cryopreservation (group A) and cryopreservation following short-term culture (group B), respective ly. LDH release, viability, protein and urea syntheses, G-6- Pase activity and diazepam transformation of the hepatocytes cultured in serum-free medium after thawing were observed during 5 days. These indices in freshly isolated and cultu red hepatocytes served as a control (group C). RESULTS:Lower viability (group A, 96.1%±19.4%; group B, 95.8%± 20.0% vs group C, 98.9%±10.3%, P<0.05) protein synthesis (group A, 36.7 ±2.2μBq·cell-1; group B, 34.8±1.9μBq ·cell-1 vs group C, 38.5±2.7μBq·cell-1, P<0.05) and diazepam transformation(diazepam concentration in group A, 7.42± 0.34mg·L-1 group B, 7.53±0.32 mg·L-1 vs group C, 6.47±0.20 mg·L-1, P<0.05) of thawed hepatocytes compared to control group were obtained in both of cryopreservation groups. There were no significant differen ces in LDH release, viability, protein synthesis, G-6-Pase activity and diazep am transformation between two cryopreservation groups. Urea synthesis in group A (3.36±0.29nmol·cell-1) was lower than in group B (3.70±0.12 nmol·cel l-1). CONCLUSION:The protocol of immediate cryopreservation of hepato cytes in RPMI1640 medium containing 100mL·L-1 DMSO, hormone, growth fact ors and newborn bovine serum with the rate controlled freezing program and rapid thawing provides indices of viability and functions of hepatocytes in subsequen t serum-free culture comparable to hepatocytes cryopreserved following short-t erm culture except for lower urea synthesis.

关 键 词：猪肝细胞 生物人工肝 暴发性肝衰竭 FHF 冷冻保存 

分 类 号：R575[医药卫生—消化系统]