提高冻存胚胎胰岛移植效果的实验研究

Experimental study on the quality and activity of fetal islets after cryopreservation

目的探讨三维组织细胞旋转培养系统(RCCS)对胚胎胰岛冻存复苏后质量的影响。方法将胚胎胰岛平均分为3组,实验组1、2为胚胎胰岛冻存前后分别用RCCS培养和普通培养,对照组新鲜胰岛经RCCS培养。切取胚胎胰腺,胶原酶V消化,纯化。然后进行标准冻存步骤,复苏后继续培养。并检测各组胰岛数量、活性、胰岛素刺激实验结果。结果纯化后收获胰岛最多每个胚胎5012.73IEQ,最少2432.68IEQ,平均(3548.07±273.46)IEQ。微重力培养组胰岛细胞存活率、胰岛素释放量、胰岛素刺激指数等均高于普通培养组。移植经过微重力培养的(2000±1)%IEQ新鲜胚胎胰岛或冻存胚胎胰岛在移植后1周内可达100%纠正糖尿病。结论微重力旋转培养有利于胰岛细胞的生长繁殖,使胰岛具有更好的胰岛素分泌能力,该方法同胰岛冻存相结合,可以进一步提高胰岛的冻存效果,为胰岛库的成功建立探索出一条新的途径。

Objective To study the impact of rotary cell culture system （RCCS） on the quality and activity of fetal islets after cryopreservation and resuscitation. Methods The fetal islets were divided into three groups averagely. In experiment groups 1 and 2, before and after cryopreservation, fetal islets were cultivated in RCCS or common culture. While in control group, fresh fetal islets were cultivated in RCCS all the time. The fetal pancrease was resected, digested with Collagenase V and purified with Ficoll solution. Then standard cryopreservtion step was adopted, and after resuscitation, the islets were cultivated continuously. The quantity, quality and activity, and insulin stimulation test results in all groups were detected. Results After purification, islets obtained from one fetal pancreas reached 5 012.73 IEQ at most, 2 432.68 IEQ at least and （3 548.07±273.46） IEQ averagely. The survival rate, the insulin release and insulin stimulation index in islets incubated in RCCS were higher than those in common culture. Transplantation of （2 000±1 ） % IEQ frozen-thawed islets would restore blood glucose to normal in all recipients within one week. And all recipients maintained the normal glucose-tolerant curve throughout the cbservation time. Conclusion RCCS are in favor of the growth and proliferation of islets, and the islets cultivated in RCCS have better insulin secreting capacity. RCCS combined standard cryopreservation method may further improve the cryopreservation effect. And this would explore a new way for islet banking establishment.