双层法氧合冷保存心跳停搏大鼠肝细胞移植研究

Two-layer method cold preservation with oxygenation for non-heart-beating donor in rat hepatocyte transplantation

目的 观察双层法（TLM）氧合冷保存较UW保存能否改善心跳停搏供体（NHBD）肝细胞存活率和功能.方法 SD大鼠为供体,建立NHBD模型,NAPs大鼠为受体.根据热缺血时间（WIT）15 m/n和30 m/n分成2组;按TLM、UW分别保存3、12 h和未保存再各分5个亚组（n=5）.检测NHBD肝细胞存活率和ATP水平,观察肝细胞移植（HTx）后肝细胞形态和功能.结果 TLM3、12 h组肝细胞存活率分别显著高于UW 3、12 h组[（69.7±4.1）%和（69.1±2.0）%比（55.1±2.3）%和（53.3±2.0）%;P〈0.01];TLM 3、12 h组AlP水平分别显著高于UW 3、12 h组（3.25±0.79和3.06±0.67比2.25±0.53和1.63±0.40;P〈0.05或P〈0.01）.HTx后几乎所有时间点TLM组血清白蛋白（ALB）水平都显著高于UW组（P〈0.05或P〈0.01）.在HTx 14d后,形态学显示TLM组肝细胞保持强活力,糖原和ALB染色呈强阳性.结论 TLM氧合冷保存可显著改善和逆转NHBD肝细胞存活率和功能,减少NHBD肝细胞缺血性损伤.

Objective To explore whether the TLM oxygenated cold preservation rescues the viability and activity of the hepatocytes from NHBD rats vs UW preservation. Methods SD rots as donors and NARs rats as recipients were used. NHBD model was induced by phrenotomy. There were two major groups according to warm ischemia time （WIT）. Hepatocytes were analyzed for viabihty, ATP and function after cold preservation for hepatocytes transplantation （HTx）. Results Hepatocyets viability in TLM group at 3 and 12 h was significantly higher than in UW group at 3 and 12 h [（69.7±4.1）% and （69.1±2.0）% vs （ 55.1±2.3） % and （53.3±2.0） % ; P〈0.01]. ATP levels in TLM group at 3 and 12 h were significantly higher than in UW group at 3 and 12 h （3.25±0.79 and 3.06±0.67 vs 2.25±0.53 and 1.63±0.40; P〈0.05 or P〈0.01）. After HTx, the serum albumin levels in TLM group were significantly higher than in UW group at almost all time points （P〈0.05）. The transplanted hepatocytes remained strongly viable in morphological findings by HE on the 14th day after HTx in TLM group, sharing the positive staining for PAS and albumin. Conclusion NHBD liver preservation by the TLM was found to be capable of producing results superior to UW to hepatocytes from rats.