人胰岛的分离纯化和功能评价

Isolation, purification, and functional evaluation of human pancreatic islet CA

目的应用Ricordi人胰岛分离技术分离和纯化人的胰岛，并进行功能和安全性的评价。方法获得6例成人尸体胰腺组织供体，采用胶原酶消化法及密度梯度离心法分离和纯化胰岛，并分析胰岛的数量、纯度和活力。采用免疫荧光法分析胰岛内分泌细胞的组成和分布；检测葡萄糖刺激的胰岛素分泌和胰岛移植后的动物血糖水平。并对分离胰岛的安全性指标进行评价。结果分离纯化后的胰岛数量为（22．9±3．1）万IEQ，纯度为（59．0±8．9）％，活力为（89．0±3．0）％。免疫荧光显示，胰岛由4种内分泌细胞组成并呈正常分布。葡萄糖刺激胰岛素释放的刺激指数为8．1±4．0。胰岛移植于糖尿病裸鼠后，血糖在3d后降至正常水平并维持超过30d。分离胰岛样本的各项安全性指标在规定范围之内。结论采用Ricordi人胰岛分离技术分离和纯化的胰岛在形态、结构、数量、纯度、活力、体内外功能以及安全性方面都达到临床胰岛移植的标准，为开展临床胰岛移植奠定了基础。

Objective To isolate and purify human islet according to the method established by Ricordi and to evaluate the function and safety of these isolated human islets. Methods Six pancreases were obtained from human corpses. The islets were isolated by liberase digestion and purificated by Ficoll density gradient centrifugation. The numbers,purity and vitality of the islets were analyzed. The various endocrine cell composition and distribution of the islets were checked by immunofluorescence staining. The glucoseinduced insulin secretion was detected by chemiluminescence method. The isolated islets were transplanted under the left renal capsules of 10 streptozocin-induced diabetic nude mice. Twenty days later the left kidneys with transplanted islets of 2 mice with normal blood sugar were resected, and then blood sugar level was observed. An isolated human islet was suspended in RPMI-1066 culture medium for 72 h, then culture of pathogenic micro-organisms, endotoxin and precoagulant activity were detected so as to evaluate the security of the islet products. Results The mean number of the isolated islets was （ 229 000 ± 31 000） islet equivalents （IEQs）/pancreas or （4970 ± 1620） IEQs/g pancreatic tissue, the mean purity was （59.0± 8. 9） %, and the mean vitality was （89 ±3 ） % for the purified islets. Immunofluorescence staining showed that there were 4 types of endocrine cells normally distributed in the islets. The mean insulin stimulation index was 8. 1 ±4. 0（3. 8 - 10. 2）. The glycemia found in the diabetic nude mice decreased to normal levels from the third day after islet transplantation and maintained normal for over 30 days. The parameters of security in these islet products were under the standard scope. Conclusion Human islets obtained according to Ricordi＇s method reach the standard for clinical islet transplantation in number, purity, vitality, function, and security.