肝细胞移植实验研究中大鼠肝细胞的冻存与复苏初探  

Study on the isolation,culture,cryopreservation,and thawing of rat hepatocyte in hepatocyte transplantation experimental research

作　　者：李力[1] 滕皋军[1] 

机构地区：[1]东南大学医学院附属中大医院放射科,南京210009

出　　处：《中华放射学杂志》2002年第4期369-372,共4页Chinese Journal of Radiology

摘　　要：目的　以大鼠为实验对象 ,研究冷冻复苏及降温速率对肝细胞活率、形态和功能的影响。方法　改良Seglen门静脉 下腔静脉双插管胶原酶灌注法分离获取游离肝细胞。分别将肝细胞按B程序 (渐进式降温程序 :- 1 5℃ /min至 - 18℃ ,保持 5min ,- 2 0℃ /min至 - 80℃ ,投入液氮保存 ) ;C程序 (慢速线形冷冻程序 :- 2 5℃ /min至 - 80℃ ,投入液氮保存 )进行处理。 2组冻存细胞分别在液氮中冷藏 3、15、30d后 ,40℃水浴复苏。检测复苏肝细胞活率、形态结构、蛋白质合成功能的改变。结果　 (1)B组、C组冻存后各时间点细胞活率及 3H 亮氨酸合成量较新鲜组 (A组 )均有明显下降 (P <0 0 0 1) ,C组明显低于B组 (P <0 0 0 5 ) ,2组各组内在不同时间点的活率及 3H 亮氨酸合成量无明显差别 (P >0 0 5 )。 (2 )光学显微镜下见复苏细胞与新鲜细胞无明显区别。电子显微镜下可见B、C组细胞超微结构有明显改变 ,但B组程度较C组轻。结论　降温速率的快慢直接关系冻存肝细胞的效果。冷冻复苏过程使细胞膜完整性受损、细胞质内容丢失导致细胞在复苏后活率降低、细胞功能丧失。肝细胞在液氮中保存 ,其活率及蛋白质合成功能并不随保存时间的延长而降低。Objective To study the effects of cryopreservation and two different cooling rates on rat hepatocytes. Methods Perfusion with collagenase digestion was used to isolate rat hepatocytes. Hepatocytes were incubated with a freezing medium consisting of RPMI 1640 with 20% calf serum and 10% DMSO. Two different freezing protocols were applied using a computer controlled freezer to freeze the medium to -80℃ before hepatocytes were plunged into liquid nitrogen and stored. The viability, morphology, and protein synthesis were measured at Day 3, Day 15, Day 30, respectively after thawing. Results Both viability and protein synthesis ability of group B and group C decreased significantly as compared with that of Group A ( P <0.001) after storage in the liquid nitrogen at intervals of 3, 15, 30 days, respectively. The results of Group B were better than that of Group C ( P <0.005). The hepatocytes in Group B and Group C did not show photo microscopically detectable alterations in cell morphology after thawing( P >0.05). Electron microscopy showed ultrastructural changes between fresh and thawed rat hepatocytes including injury of cell membrane, loss of cell content, alteration of nucleolus. The degree of injury in Group B is slighter than in Group C. Conclusion Cooling rate of cryopreservation correlates with the quality of thawed hepatocytes. The cryopreservation and thawing procedures are key factors to affect the quantity and quality of hepatocytes. The viability and protein synthesis ability are not influenced by the storage period in liquid nitrogen.

关 键 词：肝细胞移植 细胞分离 细胞存活 低温保存 降温速率 

分 类 号：R657.3[医药卫生—外科学]