Gene transfer technique for clinical islet transplantation

研究1.大鼠胰岛的基因转移。从F344大鼠分离胰岛，并在基因转移前培养3-7天。使用多胺转染试剂进行基因转染。载体为pZeoSV 2/lacZ。转基因胰岛存活5天，内分泌功能良好。X-gal染色法检测基因转移率> 90%。研究2.犬胰岛的基因转移。采用基因枪、电穿孔和脂质体转染法将PCMS-EGFP导入Beagle犬胰岛。脂质体转染技术操作简单，对胰岛损伤小。此外，脂质体转染技术可以很好地保持胰岛素的释放。研究3.临床胰岛分离、冻存和移植。在我们的研究所进行了八个人胰岛分离，使用一个脑死亡的供体和七个非心脏跳动的供体。胰岛产量为18693 ~ 491040 IEq。纯度为30 - 80%。其中一株用于临床胰岛移植。移植后，低血糖消失，血糖水平得到稳定控制。

Study 1.Gene transfer into rat islets. Islets were isolated from F344 rat and cultured for 3-7 days before gene transfer. Gene transfection was made using polyamine transfection reagents. Vector was pZeoSV2/lacZ. Gene transferred islets survived for 5 days with good endocrine function. Gene transfer rate evaluated by X-gal staining showed >90%. Study 2.Gene transfer into canine islets. PCMS-EGFP was transferred into the islats from Beagle pancreata using 1)Gene gun, 2)Electroporarion, and 3)Lipofection. Lipofection technique was simple and less harmful for the isles. Moreover, insulin release was well preserved with Lipofection technique. Study 3.Clinical islet isolation, cryopreservation, and transplantation. Eight human islet isolations were performed in our institute using one brain-dead donor and seven non-heart beating donors. Islet yield was from 18693 to 491040 IEq. Purity was from 30 to 80 %. One of these isolations were used for clinical islet allograft. After transplantation, hypoglycemia disappered and obtained stable control of blood sugar levels.