大鼠乳鼠皮肤成纤维细胞的分离和体外培养

Separation and culture of skin fibroblasts of newborn rat in vitro

目的建立大鼠乳鼠背部皮肤组织和尾尖部皮肤组织成纤维细胞的体外培养方法,并比较二者的区别。方法分别取大鼠乳鼠的背部和尾尖部皮肤组织,利用组织贴块法分离和培养皮肤成纤维细胞,用酶消化法进行细胞传代培养,采用梯度温度冻存细胞。倒置显微镜下观察成纤维细胞的形态,用细胞骨架波形蛋白抗体进行鉴定。结果光学显微镜下3-5 d可见细胞从组织块游出,7 d细胞数量增多,10 d可贴满培养瓶壁;且大鼠乳鼠背部和尾尖部来源的皮肤成纤维细胞波形蛋白表达均为阳性。结论大鼠乳鼠背部和尾尖部来源的皮肤成纤维细胞的生物特点无差别,均可作为皮肤成纤维细胞的取材部位。

Objective To establish the method of culture in vitro of skin fibroblasts which were separated from back and tail skin tissue of newborn rats. Methods The skin tissue of back and tail of newborn rats were taken and the fibroblasts were separated and cultured by explant method. The primary cultured cells were subcultured by enzyme digestion. The cultured skin fibroblasts were frozened by gradient temperature method. The cellular morphology was observed by inverted electron microscope. And the immunohistochemical staining was performed to observe Vimentin in fibroblasts. Results Several cells near the tissues could be visible 3- 5 days after tissues were explanted,and the cell number was increased on the 7^th day and the cells could be covered with flask wall on the 10 thday. The skin fibroblasts from back and tail of the newborn rats were found to express Vimentin by immunofluorescence staining. Conclusions There is no difference between the cultured skin fibroblasts derived from back and tail of the newborn rats. Both of the back and tail skin tissue of newborn rats can be used as the material of skin fibroblasts culture in vitro.