腹主动脉灌注法分离胰腺星状细胞及其培养、鉴定

Isolation of pancreatic stellate cells using collagenase perfusion method through abdominal aorta cannulation and its culture and characterization

目的:通过腹主动脉插管灌注胶原酶分离胰腺星状细胞(pancreaticstellatecell,PSC),建立简单易行的PSC分离方法,并对PSC进行培养和鉴定。方法:大鼠腹主动脉插管灌注后,经胶原酶消化、Nycodenz密度梯度离心,分离得到PSC;通过观察细胞形态、胞质内脂滴和免疫细胞化学方法检测结蛋白(desmin)、神经胶质酸纤维蛋白(glialfibrillaryacidicprotein,GFAP)、α平滑肌肌动素(α-smoothmuscleactin,α-SMA)的表达来鉴定PSC。结果:腹主动脉灌注法分离得到PSC,产率、活力和纯度分别为(:15.3±4.6)×103/g体重(﹑95.0±3.5)%﹑﹥80%;培养24h后大多数细胞已贴壁,呈星状或多角形,48h后表达desmin、GFAP,并在96h后表达α-SMA。结论:通过腹主动脉灌注法较容易分离得到PSC,其产率、活力和纯度较高,经鉴定后能够满足体外实验要求。

Objectlve：To establish a simple collagenase perfusion method through abdominal aorta cannulation to isolate rat pancreatic stellate cell and demonstrate the procedures of its culture and characterization. Methods ：Via a cannula iserted into adominal aorta, Gey＇s balanced salt solution containing 0.025% collagenase P was perfused into rat normal pancreas. After digestion with collagenase P and protease, Nycodenz density gradient centrifugation was performed to isolate PSC. PSC was identified by its morphology, cytoplasmic lipid droplets and immunocytochemical staining for desmin, GFAP and α-SMA. Results：The production, viability and purity of isolated PSC were （15.3 ± 4.6） × 10^3/g body weight, （95.0 ± 3.5）%, and 〉 80% respectively. After 24 hours in culture, most PSC attached to culture dishes, and showed angular or stellate appearance. PSC became positive for desmin and GFAP after 48 hours in culture but did not express α-SMA until 96 hours in culture. Conclusion ：The collagenase perfusion method through abdominal aorta cannulation could be used for PSC isolation, with high production, viability and purity, therefore, it could to meet the need of in vitro experiments.