【作者】 何波；

【导师】 王增涛；

【作者基本信息】 山东大学 ， 外科学， 2008， 硕士

【摘要】 研究背景:近年来,深低温保存技术的应用越来越广泛。深低温可以降低细胞的呼吸代谢作用,延缓其功能的丧失,临床上将生物组织或器官采用特殊方法冷却至深低温,并长期保存;待需要时,再将其按特殊方法复温,以便获得活的生物体。2002至2006年,我们先后将2例患者的断指在冷冻保护剂二甲亚砜(DMSO)的作用下,通过程序降温在-196℃液氮中保存后再植成功。但术后长期随访发现,患指均有不同程度萎缩。据冷冻损伤的两因素假说指出,造成细胞损伤有两个独立因素:胞内冰损伤和溶质损伤。一是胞内冰的形成,由过快冷却所产生的;冷却速率越快,此损伤越大。另一是“溶液损伤”,由过慢冷却所产生,使细胞在高浓度溶液中暴露时间过长而遭损伤,冷却越慢,此损伤越大。但是,目前对低温损伤两因素引起细胞的死亡方式没有进行阐明。为阐明低温损伤两因素引起细胞的死亡方式,我们选取人肝癌细胞株BEL-7402经荧光染色后流式细胞仪检测细胞凋亡、坏死、和细胞碎片的变化。研究深低温冻存导致细胞死亡的方式,并观察对细胞周期的影响。目的研究深低温冷冻损伤导致细胞死亡的机制。方法在不同的电解质与冷冻保护剂浓度下:1倍离子浓度下50%甘油、0%、10%、50%DMSO及20倍离子浓度下50%DMSO,对人肝癌株细胞株(BEL-7402)程序降温至深低温后复苏。采用Annexin-V/PI荧光标记双染色、DNA-Prep stain荧光标记染色流式细胞仪分析冻存前后凋亡、坏死、存活、细胞碎片及BEL-7402细胞周期变化。结果无冷冻保护剂时,冷冻后细胞坏死、碎片明显(P＜0.05)。随着离子浓度和DMSO浓度的增加,冷冻后BEL-7402细胞凋亡愈趋明显,但细胞坏死、碎片均不明显(P＞0.05);冷冻前后细胞周期变化不显著(P＞0.05)。结论在冷冻过程中,造成细胞损伤有两个独立因素:胞内冰损伤和溶质损伤。胞内冰损伤引起细胞死亡的方式是坏死,而溶质损伤(包括电解质与DMSO)导致细胞凋亡。冷冻导致细胞死亡无周期特异性。更多还原

【Abstract】 Background During the past ten years,biologists had found the method to cryopreserve many biomaterials,such as sperms,skin,parathyroid gland,leukocytes, bone marrow cells and antibiotics.Report showed that a cryopreserved Cyprinus Carpio embryo was still alive after thawing.In 2002,we succeeded in reimplanting a disjunct finger which was thermal-control cryopreserved in liquid nitrogen for 81 days.This success provided us a perspective in the cryopreservation of organs,or even the whole body of patients suffering from cancers.To investigate influence of cryodamage on cell viability,a study on mechanism of cryodamage in human hepatoma carcinoma cell was carried on.Objective To investigate influence of cryodamage on cell viability.Methods Human hepatoma carcinoma cell line BEL-7402 were cryopreserved under different concentrations of electrolytes and cryoprotective agents:1×50% glycerine,and 0%,10%or 50%DMSO;or 20×50%DMSO.Then apoptosis,cell necrosis,cryosurvival and debris rates of the cells,and cell cycle of BEL-7402 before and after freezing were analyzed by flow cytometry.Results Without cryoprotective agents,cell necrosis and debris were obviously observed(P＜0.05)after freezing.When the concentrations of electrolytes and DMSO were increased,cell necrosis and debris got diminished(P＞0.05),while apoptosis of BEL-7402 turned to be significant gradually;There were no significant variation of cell cyle after freezing(P＞0.05). Conclusion There are two independent kinds of cryodamage factors:one is endocelluar cryodamage,which can bring about cellular necrosis,and the other is solute-related damage,which may induce apoptosis.Both of these two kinds of cryodamage have no cyclic specificity.更多还原