成人胰岛细胞的分离与纯化

Isolation and purification of pancreatic islets in adult donors

目的 探讨体外分离与纯化成人胰岛细胞的方法与可行性，为胰岛细胞移植治疗1型糖尿病提供大量高质量的胰岛细胞。方法获取的成人胰腺组织称重后，采用V型胶原酶消化法分离；再用Ficoll间断密度梯度离心纯化法纯化；在DTZ染色显微镜下，评价胰岛细胞的数量、纯度；并用体外培养、放射免疫测定胰岛素法鉴定胰岛细胞活性。结果成人胰腺经胶原酶消化分离后，胰岛平均收获量（3600±447）个／g胰腺；Ficoll间断密度梯度离心纯化后，胰岛平均收获量（2140±207）个／g胰腺，纯度〉70％；成人胰岛细胞培养2、4、6d后，测定其培养液中基础胰岛素浓度（mIU·L^-1·100^-1）分别为（3．302±1．63）、（3．504±1．10）、（2．921±1．13）。结论胶原酶消化法、Ficoll间断密度梯度离心纯化法分离纯化成人胰岛是有效的方法。

Objective To investigate the methods and feasibility of islets isolation and purification, and to get more islets with high purity and good function for clinical transplantation. Methods After being weighted, human pancreatic islets were isolated with type V collagenase, and purified by Ficoll＇s discontinuous density gradient centrifugation. The yield and purity of islets were evaluated by dithizone（DTZ） staining under microscope, and the viability was assessed by insulin release assay in vitro. Results The average number of islets was about 3 600 ±447 per gram pancreas after isolation and it was about 2 140±207 after purification with more than 70% purity. 2, 4, 6 days after islet cell culture, the basal insulin concentration of the culture medimu was measured, and it was 3. 302 ±1.63, 3. 504±1.10, and 2. 921± 1.13 （mIU/L/100 islets） respectively. Conclusion Collagenase digest and Ficoll＇s discontinuous density gradient centrifugation are effective methods for isolation and purification of human pancreatic islets.