成人胰岛大容量分离技术的改进及胰岛功能检测

目的 通过对人胰岛分离技术的改进以获得大量高活力胰岛并检测其功能,为利用同种异体胰岛移植治疗1型和部分2型糖尿病提供理论依据和技术基础。方法 采用改良的自动分离技术连续分离28例人胰岛,然后用连续性密度样度离心法纯化胰岛。胰岛收获量以国际标准的胰岛当量(islet equivalent,IEQ)表示。胰岛功能的测定分别为体外测定胰岛的胰岛素／DNA比率;静止葡萄糖刺激试验(SGS)及将胰岛移植至糖尿病裸小鼠的体内胰岛功能鉴定并随后进行腹腔糖耐量试验,连续测定移植鼠血糖水平及其体内C肽浓度。结果 28例成人胰腺分离的胰岛收获量为5000～1030000IEQ／胰腺,平均为291635IEQ／胰腺,前13例平均每个胰腺收获49123IEQ,平均每克组织收获846IEQ。平均纯度为87％,随着技术的改进后15例的分离结果则分别为：平均每个胰腺501813IEQ,平均每克组织7003IEQ,平均纯度89％。体外胰岛素刺激试验结果表明分离纯化后的人胰岛有正常功能,将12次分离得到的胰岛分别移植至34只糖尿病裸鼠肾包膜下,其中29只糖尿病裸鼠于12h内血糖恢复正常且糖耐量试验接近正常鼠,血中C肽水平亦接近正常鼠。结论 采用改进的人胰岛分离方法,可以获得大量高活力的具有正常功能的胰岛,为同种异体胰岛移植用于临床奠定了必要的实验基础。

Improvement of massive human islet isolation techniques and the evaluation of isolated human islets

OBJECTIVE: To obtain massive human pancreatic islets with modified techniques and evaluation of the islets for the clinical allo-transplantation to treat type I and II diabetes. METHODS: 28 consecutive adult human pancreata were isolated with modified automated techniques. Islets were purified using continuous density gradient. The islet yield was counted with international standard known as islet equivalent (IEQ). The function of the isolated islets was evaluated by measuring DNA/insulin ratio, static glucose stimulating test in vitro and transplanting the islets into diabetic nude mice in vivo followed by abdominal glucose tolerance test and C peptide measurement. RESULTS: The yield of 28 consecutive human pancreata isolations ranged from 5 000 to 1 030 000 IEQs/pancreas with the average of 291 635 IEQs/pancreas. The first 13 isolations yielded 49 123 IEQs/pancreas, 846 IEQs/g and, purity 87% in average. The remained 15 isolations after the modifications yielded 501 813 IEQs/pancreas, 7 003 IEQs/g and purity 89% in average. The results of in vitro SGS showed good response to the different glucose concentration. 34 diabetic nude mice were transplanted under the renal capsule with the freshly isolated islets. 29 out of 34 diabetic mice obtained normoglycemia within 12 hours and the glucose tolerance tests were near normal. Serum C peptide level of transplanted mice is close to that of the control group. CONCLUSIONS: Massive human islets can be isolated with the modified techniques. Quality assessment of these islets both in vitro and in vivo has indicated that these high quality human islets could be used for the clinical allogeneic islet transplantation.