大鼠胰星状细胞的分离与培养

Isolation and culture of rat pancreatic stellate cells

目的 建立大鼠胰星状细胞 (pancreatic stellate cells,PSCs)的分离培养方法。方法 大鼠胰腺组织经胶原酶和链霉蛋白酶 E消化后 ,用 Nycodenz不连续密度梯度离心法分离 PSC,在 32 8nm紫外光激发下观察细胞的自发荧光现象 ,并以免疫组化技术检测结蛋白 (desmin)、神经胶质原纤维酸性蛋白 (glial fibrillary acidic protein,GFAP)和α-平滑肌动蛋白 (α- sm ooth m uscle actin,α- SMA )的表达 ,同时观察培养细胞的形态和生长特性。结果 新鲜分离的大鼠 PSCs产率、活力和纯度分别约为 2 .5 ×10 6 /g胰腺、95 %和 90 %。培养的 PSCs可自发活化 ,表达 α- SMA,细胞由静止型转化为肌成纤维样细胞表型。原代培养 10天后细胞纯度 >95 % ,传代培养后细胞纯度可达 99%以上。结论 利用 Nycodenz密度梯度离心方法可成功分离大鼠 PSCs,其细胞产率、活力和纯度均可满足体外研究需要。

Objective To establish procedures for isolation, culture, and identification of rat pancreatic stellate cells (PSCs). Methods PSCs were isolated from normal rat pancreas using Nycodenz discontinuous density gradient centrifugation following digestion with collagenase and Pronase E, and the purity was evaluated by autofluorescence phenomenon at the wavelength of 328 nm under a laser confocal scanning microscope and immunocytochemical expression of desmin, GFAP, and α-SMA. Phase contrast morphologic examination was also performed to observe cell phenotype and growth characteristics. Results The production, viability, and the purity of freshly isolated PSCs were about 2.5 × 106/g pancreas, 95%, and 90%, respectively. During primary culture, the cells were activated, changing from a quiescent fat-storing phenotype to a myofibroblast-like cell expressing α-SMA ( > 95% when cultured for 10 days, and > 99% after one passage). Conclusions Using a discontinuous gradient of Nycodenz, rat PSCs can be isolated with very high production and purity to meet the need of in vitro investigations.