冷缺血时间及血液相容性与移植胰岛细胞活性的关系

背景:在胰岛移植过程中,胰岛细胞的数量和胰岛细胞的活性是非常重要的。胰岛细胞移植中冷缺血时间和组织相容性是关系胰岛细胞移植能否成功的关键问题。目的:观察冷缺血时间及血液相容性不同对于胰岛细胞数量和活性的影响。设计:观察性实验。单位:广西中医学院附属瑞康医院。材料:采用脑死亡自愿捐赠者的器官(已知血型和HLA配型),在其他器官切取后或同时进行胰腺的获取;血型相同、HLA配型相符的血液;HLA配型高致敏(错配3个以上位点)或群体反应性抗体>50%或淋巴毒阳性受者的血液;分离纯化后的胰岛细胞悬浊液,经过70μm的筛网滤过后,保留直径大于70μm的胰岛细胞,配置成浓度1.20×105L-1左右活性胰岛细胞悬浊液。方法:高渗枸橼酸盐嘌呤溶液经过腹主动脉原位灌注后,参照文献进行肝、肾、胰腺联合及分别切取。用于切取的时间:胰腺肾脏的联合切取、肝脏胰腺肾脏的联合切取方法平均需时15min,肝脏、胰腺、肾脏的单独切取平均需时20min,用于胰岛分离的胰腺12份,冷缺血时间2.5～8h。冷却血时间在5h以内的9份,温缺血时间0～3min,消化的时间15±2.4min。采用胶原霉P进行消化分离胰岛细胞,双硫腙、丫腚橙染色测定不同的冷缺血时间条件下活性胰岛细胞活性,分析体外胰岛细胞和血液与供受者的组织相容性和胰岛细胞存活的关系。主要观察指标:胰岛细胞活性率和血小板、中性粒细胞和单核细胞计数。结果:肝、肾、胰腺联合切取及各器官的单独切取顺利,在冷却血5h以内胰岛细胞活性率都在80%以上,用于胰岛细胞移植的胰腺和其他器官的切取不会影响胰岛细胞的活性;人类胰岛暴露于未经抗凝的人类血中,胰岛将诱发一个迅速的血细胞消耗;进行血小板、中性粒细胞和单核细胞计数,HLA错配组和HLA匹配组与对照组比较血细胞都发生明显的消耗;加入肝素后血细胞计数与对照组比较差异明显(P<0.05),反应明显减轻。HLA匹配组和HLA错配组胰岛细胞体外培养24h活性胰岛细胞数量较,差异明显(P<0.05)。结论:在冷缺血时间<5h的情况下获取的胰腺可以用于临床胰岛细胞的移植;血液相容性好能够明显提高胰岛细胞移植的成功率。

Cold ischemia time and blood compatibility associated with activity of transplanted islet cells

BACKGROUND: The quantity and bioactivity of isolated islet are vital to islet transplantation; while, the cold ischemia time and human leucocyte antigen (HLA) typing are key factors to islet quantity and bioactivity which inflect islet transplantation.OBJECTIVE: To observe the effects of cold ischemia time and blood compatibility on quantity and bioactivity of islet cells.DESIGN: Observational study.SETTING: Ruikang Affiliated Hospital of Guangxi College of Traditional Chinese Medicine.MATERIALS: Organs from voluntary donors died of irreversible coma were adopted whose blood-type and HLA typing had been known, pancreases acquisition was carried on after other organs ablation or in the meantime. The blood of identical ABO and HLA matching conformity, or HLA cross-matching hypersensitization (missmatching over 3 Iocuses),or panel reaction antibody (PRA) ＞ 50%, or lymphocyte cytotoxin crossmatching test positive. The isolated and purified islet suspension was filtered by 70 μm filter, which result in preparing 1.2×105/L islet suspention.METHODS: Hypertonic citrate adenine solution was perfused into aorta, and kidney-pancreases and kidney-pancreases-liver were cut together or kidney-pancreases-liver was cut separately. Islet activity was judged by diphenylthiocarbazone (DTZ) dyeing and acridine orange (AO) dyeing; meanwhile, twelve pancreases far from contamination were aquired, mean ablation time was 15 minutes; cold ischemia time ranged from 2.5 to 8 hours. Cold ischemia time of nine pancreases was controlled in 5 hours, warm ischemia time was 0-3 minutes, and peptic time was (15±2.4) minutes, correlation of islet cells and histocompatibility with survival of islet cells was analyzed.MAIN OUTCOME MEASURES: Survival rate of islet cells; counts of platelet, heterophil granulocyte and monocyte.RESULTS: The cutting of kidney, pancreases and liver were successful. If the cold ischemia time was controlled within 5 hours, activity of islets was above 80%. Pancreatic gland used for islet transplantation and cutting of other organs could not affect activity of islet cells. When human islets were exposed to human blood, it would induce a rapid consumption of platelets, neutrophils, monocytes and lymphocytes in the blood. Consumption of blood cells was more in the HLA typing groups than that in the control group. After adding heparin, there was significant difference in cell account among the three groups (P ＜ 0.05) and the reaction was relieved obviously. After 24-hour cultivation, there were significant difference in active islets quantity between HLA typing compatibility group and HLA typing incompatibility group (P ＜ 0.05).CONCLUSION: Pancreatic gland obtained under the cold ischemia time ＜ 5 hours can be used in clinical transplantation of islet cells; a good histocompatibility can raise successful rate of transplantation of islet cells.