胰岛移植物质量鉴定胰岛移植物质量鉴定.docVIP

胰岛移植物质量鉴定方法的研究 摘　要　胰岛移植物的质量直接影响胰岛移植的效果，用经胶原酶消化、Ficoll纯化的成年大鼠胰岛，比较乙醇和二甲亚砜(DMSO)配制的双硫腙(DTZ)，作B细胞鉴别染色；丫啶橙(AO)-碘丙啶(PI)和双醋酸荧光素(FDA)-溴乙锭(EB)双色荧光染色，鉴别活细胞。结果表明，乙醇配制的DTZ染色对细胞毒性小，且染液易储存；AO-PI染色，因AO能识别凋亡细胞，且着色鲜明，荧光持久。采用DTZ和AO-PI联合染色，能对胰岛的纯度、数量和活率作出精确的鉴定。　　关键词　胰岛质量　　双硫腙　　丫啶橙　　碘丙啶 Methods for evaluation of the islet graft quality 　Dong Weiping, Zhang Hongde, Wang Yufei, et al. Diabetes Research Laboratory, Shanghai First Peoples Hospital, Shanghai 200080　　Abstract　The identification of beta cell and living cells of purified islets of adult rats were comparatively studied with ethanol or DMSO prepared dithizone (DTZ) and with acridine orange (AO)-propidium iodide (PI) or fluorescent diacetate (FDA)-ethidium bromide (EB) respectively. The results indicated that the function and viability of islet cells were not influenced by the staining, the insulin content of the culture medium and the insulin release test of the stained islet were not significantly different from those of the unstained controls (P＞0.05). The cell-toxicity of ethanol prepared DTZ was much less than that of DMSO prepared. In addition, the ethanol prepared DTZ was easily preserved. AO-PI staining could identify the apoptotic cells because the binding of AO with double chain DNA showed green fluorescence, while the binding of AO with single chain DNA (degenerated DNA) showed red fluorescence. Therefore, DTZ and AO-PI combined staining may exactly identify the purity, amount and viability of the islets.　　Key words　Islet quality Dithizone Acridine orange Propidium iodide 　　临床胰岛移植的效果目前还不理想，主要由于移植物的质量与数量不足和移植物的免疫性排斥。本文报道我们近来对胰岛移植物质量鉴定方法的研究结果。 材料与方法　　一、材料　　1.动物：成年SD大鼠(上海计划生育研究所提供)。　　2.试剂：胶原酶(Ⅴ型)；双硫腙(DTZ)；丫啶橙(AO)；碘丙啶(PI)；双醋酸荧光素(FDA)；溴乙锭(EB)；二甲亚砜(DMSO)，以上试剂均系美国SIGMA公司产品。　　二、方法　　1.大鼠胰岛制备：成年大鼠经戊巴比妥钠麻醉，消毒，剖腹，放血处死，胆总管原位插管，注入预温的0.5g*L-1胶原酶溶液(含7.5mmol*L-1Ca++，10mmol*L-1HEPES，pH7.8)8ml，分离已膨胀的胰腺，38℃水浴振荡10分钟，取出，于冰浴中撕碎，移入消化瓶中，38℃水浴振荡5分钟，取出振摇，使呈细沙状，20目不锈钢丝网过筛，加入冰冷的Hanks液(含10%新生牛血清NCS)，终止消化，4℃，1000转离心3分钟，弃上清，同法洗二次，沉淀，加25%Fico