家兔胰岛分离纯化方法的改进

An improved method on isolation and purification of rabbit islets

目的介绍一种新的家兔胰岛分离纯化方法并进行纯化后胰岛的外观、活性和胰岛细胞团表面结构等质量分析。方法雄性日本大耳白,采用2 mg/m L胶原酶V逆行胰管灌注和消化,并用Hanks液洗涤,用Histopaque-1119、Histopaque-1077密度梯度离心进行胰岛纯化,DTZ、FD-PI染色鉴定胰岛纯度和活性,扫描电镜观察纯化后胰岛表面结构。结果家兔胰管开口于小肠,纯化得到家兔胰岛外形圆整,纯度>90%,活性(87.4±5.9)%,纯化前胰岛当量为(1 874.8±464.5)IEQ,纯化后胰岛当量为(1 254.8±235.8)IEQ;扫描电镜结果显示胰岛细胞团表面有被膜,但部分胰岛被膜被消化。结论采用此方法可快速得到纯度高、活性好且结构完整的家兔胰岛,为进一步进行家兔胰岛体外质量及体内移植研究奠定了基础。

Objective An improved method about how to isolate and purify rabbit islets was introduced, and its morphology, activity, and the outer surface structure of the purified islets were evaluated. Methods Male Japanese white big-ear rabbits were chosen in this experiment, 2 mg/m L collagenase V was perfused into the pancreatic duct. Then the pancreas was digested and washed by Hanks solution for three times. The tissues were purified by centrifugation with discontinuous density gradient： Histopaque -1119 and Histopaque -1077. DTZ,FD-PI staining were done to identify islet purity and activity. Scan electron microscope inspection was done to evaluate the outer structure of the islet. Results Rabbit pancreatic duct opened to the small intestine. The islets were round, and the purity was 90%, the activity was（87.4 ± 5.9）%.（1 874.8 ± 464.5） IEQ was got before purification and（1 254.8 ± 235.8） IEQ after purification islets from rabbits. The results of scanning electron microscopy showed that there was a membrane cover the surface of rabbit islet, but it was likely to be digested.Conclusion High purity, activity and intact islets quality can be got from the rabbit by this method, which makes a good foundation for the further quality research of islet in vitro and the islet transplantation in vivo.