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目的探讨胰岛细胞的分离、培养与鉴定。方法从实验室中挑选出30只成年雄性大鼠,将胶原酶注入大鼠的胰腺导管,聚蔗糖梯度离心法分离胰岛细胞,并培养与鉴定胰岛细胞。培养1周后用双硫腙(DTZ)行胰岛细胞鉴定。用丫啶橙(AO)、碘丙啶(PI)检测胰岛细胞活性。结果经过分离与纯化,大鼠胰岛细胞的获得率较高且较稳定;分离后的胰岛细胞在适宜环境中生长,生长状态较佳,在培养后的12~24 h可贴壁,在培养后4~5 d细胞生长状态达到顶峰,细胞完好,且折光性能优异。利用免疫组化法鉴定胰岛细胞,分离的细胞SMA及PDGFR表达呈阳性,少数细胞v WF表达呈阳性。结论我科此次分离纯化大鼠胰岛细胞的方法为逆行灌注胶原酶+原位消化+Ficoll液梯度分离法,实验结果显著,能在一定程度上获取纯度较高的大鼠胰岛细胞。分离后的胰岛细胞在培养4~5 d后达到最佳状态,适用于作为实验室胰岛细胞功能深入研究的实验材料。
Objective To investigate the isolation, culture and identification of islet cells. Methods Thirty adult male rats were selected from the laboratory. The collagenase was injected into the pancreatic duct of rats. The islet cells were separated by Ficoll gradient centrifugation, and the islet cells were cultured and identified. After cultured for 1 week, the islet cells were identified by dithizone (DTZ). Islet cell activity was detected by acridine orange (AO) and iodine (PI). Results After isolation and purification, the islet cells obtained in rat were higher and more stable. The isolated islet cells grew well in suitable environment and grew well 12 to 24 h after culture, and after cultured 4 ~ 5 d cell growth peaked, the cells intact, and refraction performance is excellent. The islet cells were identified by immunohistochemistry, the positive cells were found in SMA and PDGFR, while the number of vWF was positive in a few cells. Conclusion The method of separating and purifying rat pancreatic islets by retrograde perfusion collagenase + in situ digestion + Ficoll liquid gradient separation method, the experimental results were significant, to a certain extent, to obtain high purity rat islet cells. The isolated islet cells reached the best state after 4 to 5 days of culture, which is suitable for further study on the function of islet cells in laboratory.