胰岛分离、纯化制备的改进

目的 探讨大型哺乳动物胰岛机械化大量分离、纯化的方法,为人类胰岛移植物的大量制备摸索创造条件.方法 应用改进的机械化胰岛分离、纯化系统,用HCA和UW液顺序原位灌洗犬胰腺,主副胰管插管,4℃胶原酶-V(1.5 g/L)+胰酶抑制剂pefabloc(0.4 mmol/L)灌注后,Ricordi-Chamber消化罐消化,4℃COBE2991连续密度梯度离心纯化,测定胰岛当量(IEQ)、胰岛纯度及存活率、胰岛素及C-肽的释放量、培养24 h后光镜及电镜观察.结果 胰腺消化时间为(25.0±6.0)min,胰岛外分泌腺包裹率为(9.4±2.4)%,消化后胰岛收获量为(17.2±3.6)×104IEQ/每个胰腺,纯化后胰岛收获量为(8.3±2.0)×104IEQ/每个胰腺,胰岛纯度为(89.7±3.5)%.纯化胰岛体外低糖与高糖刺激下胰岛索分泌量及C-肽的释放量良好,培养24 h后形态结构及功能正常.结论 本实验室改进的胰岛机械分离方法及各设备运行可靠,获得的胰岛形态功能良好,可望用于临床人类胰岛的大量制备.

Improved automated method for isolation of canine pancreatic islets

Objective:To study an improved automated method for isolation and purification of large amounts of human pancreatic islets from large mammals, and try to create conditions for preparation and isolation of large amounts of human islets.Methods:An improved automated system was used to isolate and purify panreatic islets of sogs. Under the general anaesthesia(GA) condition, the pancreas of the dogs was via in situ vascular perfused using cold HC-A solution and then removed en bloc with duodenum and spleen. Then they were placed in cold UW aolutian. Intraductal collagenase-Ⅴ and pefabloc (4 ℃) was delivered with controlled perfusion. lslets were dissociated in system of Ricordi Chamber and purified islets separated with Ficoll density gradient centrifugation under controlled temperature. Digestion time, islets remaining trapped in exocrine tissue,final islet purity, insulin and C-pep secretory activity, and islet recovery were observed. The purified islets were observed under light microscope and electronic microscope after 24 h culture.Results:The digestion time rate was (25.0±6.0) min,islets remaining trapped in exocrine tissue was(9.4±2.4)%, final islet purify rate was (89.7±3.5)%, islet recovery after digestion was(17.2±3.6)×104 IEQ/pancreas. Islet recovery after purification was (8.3±2.0)×104IEQ/pancreas. Insulin and C-pep secretory activity of the purified islets and their ultrastructure after 24 h culture were ideal.Conclusions:Our improved automated method and facilities for isolation of canine pancreatic islets are reliable, The morphology and function of the procured islets are excellent and they can foreseeably be used for large-scale preparation of huma islets for clinical use.