人、鼠胰星状细胞3种细胞标志物desmin、GFAP、α-SMA的表达

Expression of desmin, GFAP and α-SMA in human and RAT pancreatic stellate cells

目的研究人和大鼠胰星状细胞(PSCs)在培养过程中细胞标志物表达的动态变化。方法人和大鼠胰腺组织经胶原酶、链霉蛋白酶E和DNaseI联合消化后,采用Nycodenz不连续密度梯度离心方法分离PSCs,获得的细胞采用离心淘洗技术进行纯化。用激光共聚焦扫描显微镜观察新鲜分离细胞的维生素A(VitA)自发荧光现象,用免疫组化法检测细胞标志物———结蛋白(desmin)、神经胶质原纤维酸性蛋白(GFAP)和α-平滑肌动蛋白(α-SMA)的表达。细胞接种培养后,观察细胞形态和生长特性,采用Western和Northern分析分别检测细胞标志物和I型前胶原α1链基因表达的动态变化。结果采用离心淘洗技术获得的人和大鼠PSCs纯度分别可达85%以上和95%以上。新分离的人和大鼠PSCs胞浆内有VitA自发性蓝绿色荧光。新分离大鼠PSCs表达desmin和GFAP,不表达α-SMA,而人PSCs均不表达desmin、GFAP或α-SMA。在体外培养过程中,大鼠PSCs表达desmin和GFAP逐渐减弱,但人和大鼠PSCs均开始大量表达α-SMA蛋白和Ⅰ型前胶原基因。结论利用离心淘洗技术可获得高纯度的人和大鼠PSCs。人和大鼠PSCs细胞标志物表达存在种属差异,但它们活化后均高表达α-SMA蛋白和I型前胶原基因。

Objective To investigate the dynamic changes in expression of cell markers desmin, glial fibrillary acidic protein (GFAP), and (-smooth muscle actin (α-SMA) in primary cultures of human and rat pancreatic stellate cells (PSCs). Methods PSCs were isolated from human as well as rat pancreas using Nycodenz discontinuous density gradient centrifugation following digestion with combination of collagenase IV, Pronase E and DNase I, and purified by centrifugal elution techniques. Freshly isolated cells were examined by laser scanning confocal microscopy for vitamin A autofluorescence, by immunostaining for desmin, GFAP, and α-SMA. Expression of α-SMA was as well measured by Western analysis. Procollagen α1(Ⅰ) mRNA expression was analyzed by Northern analysis. Results The purity of rat PSCs obtained by centrifugal elution were above 95%. More than 85% of either freshly-isolated human or rat PSCs displayed positive vitamin A autofluorescence. Rat PSCs stained positively for desmin and GFAP and negatively for α-SMA, whereas human PSCs were negative for either desmin, GFAP or α-SMA. During the process of primary culture, rat PSCs were positive for α-SMA at 3d and completely transformed from quiescent state to myofibroblast-like phenotypes at 7d, which negatively or scarcely expressed desmin and GFAP, but fully expressed the α-SMA protein and procollagen α1(Ⅰ) mRNA, similarly to the settings of human PSCs. Conclusions Human and rat PSCs could be successively isolated in above 95% purity by combining gradient centrifugation with following centrifugal elution techniques. The results show some species differences in desmin and GFAP expression between freshly-isolated human and rat PSCs. Both of which, however, acquire a myofibroblast-like phenotype largely expressing α-SMA protein and procollagen α1(Ⅰ) gene in culture.