肝细胞冻存复苏成活率及胆红素代谢活性的实验评价

Experimental evaluation of the survival rate of resuscitation of adult mice hepatocytes after cryopreservation and its metabolic activity to human bilirubin

目的评价成年异种动物肝细胞液氮冻存复苏后对人重型肝炎胆红素的代谢活性,探讨生物人工肝支持系统(EBLSS)使用冻存复苏异种肝细胞的实际价值。方法采用HE染色镜检肝细胞形态、锥虫兰染色计数肝细胞成活率、细胞培养检测胆红素代谢活性,实验观察液氮中冻存3个月复苏后的成年小白鼠肝细胞形态、存活率和对重型肝炎胆红素代谢活性,并设刚分离的肝细胞作对照组。结果冻存复苏后肝细胞胞质严重丢失,成活率约为30%,重型肝炎的总胆红素、直接胆红素、间接胆红素分别下降8.63%、8.98%、8.06%;新鲜分离肝细胞形态完整,成活率为90%以上,使重型肝炎总胆红素、间接胆红素、直接胆红素分别下降14.09%、12.95%、20.82%(t=8.932,P<0.01)。结论冻存复苏的异种肝细胞胞质丢失严重,细胞成活率很低,对胆红素代谢活性很弱,不是EBLSS理想的生物材料。

Objective To evaluate the metabolic activity of adult xenogeneic animal hepatocytes to bilirubin after resuscitation from liquefacient nitrogen cryopreservation of patients with severe hepatitis. Methods To determine the metabolic activity of bilirubin microscopic examination of hepatocytes by HE staining, the survival rate of hepatocytes by trypan blue staining, cell culture and Beckmarm auto -analyzer. The morphologic changes of fresh hepatocytes were regarded as control group. Results The cytoplasm of hepatocytes after cryopreservation resuscitation lost markedly, the survival rate was about 30% ,the serum bilirubin, direct bilirubin and indirect bilirubin of severe hepatitis fell 8.63%, 8.98%, and 8.06%, respectively. The morphology of fresh-isolated hepatocytes was well preserved,the survival rate was above 90%. The serum bilirubin, direct bilirubin and indirect bilirubin fell 14.09% ,12.95% ,20.82%, respectively （t = 8.932, P 〈0.01）. Conclusion The allograph hepatocytes after cryopreservation resuscitation are not ideal biological material for bioartificial liver support system because of the large lose of their cytoplasm, low survival rate and weak metabolic activity to human bilirubin.