大量人胰岛分离技术的改进及相应胰岛功能的检测

Improvement of massive human islet isolation techniques and their quality evaluation

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。方法采用经典及改良技术分离、纯化28例人胰岛。胰岛收获量以胰岛当量(IEQ)表示。胰岛功能测定分别为体外测定胰岛素/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功能鉴定并行糖耐量试验,连续测血糖水平及其体内C肽浓度。结果 前13例采用经典方法,平均每个胰腺收获49123 IEQs,平均846 IEQs/g胰腺组织,平均纯度为87％。改进技术后15例的分离结果则分别为:501813 IEQs/胰腺,7003 IEQ/g胰腺组织及纯度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。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基础。

Objective To obtain massive human pancreatic islets with modified techniques and to evaluate the function of the isolated islets. Methods Twenty-eight adult human islets of pancreas were isolated and purified with classic and modified automated techniques respectively. The islet yield was counted as islet equivalent (IEQ). The function of the isolated islets was evaluated by measuring DNA/ insulin ratio, static glucose stimulating test (SGS) in vitro and transplanting the islets into diabetic nude mice followed by abdominal glucose tolerance test and C peptide measurement. Results The first 13 isolations with the classic method 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 IEQ/g and, purity 89% in average. The results of in vitro SGS showed good response to the different glucose concentrations. Thirty-four diabetic nude mice were transplanted with the freshly isolated islets with 29 obtained normoglycemia within 12 h and the glucose tolerance tests were near normal. Serum C peptide level of transplanted mice was close to that of the control group. Conclusion Massive human islets with normal function can be isolated with the modified techniques.