人2型糖尿病皮肤角质形成细胞的无血清培养及纯化

Primary culture and purification of type 2 diabetic human keratinocytes in serum-free medium

目的体外原代无血清培养人2型糖尿病皮肤角质形成细胞,并进行纯化。方法采用中性蛋白酶-胰蛋白酶两步消化法分离人2型糖尿病皮肤角质形成细胞,使用无血清培养基进行原代培养,通过两步消化传代纯化细胞,使用倒置相差显微镜观察细胞生长状态,WST-8法测定细胞增殖曲线,流式细胞术检测其表型和纯度。结果无血清培养并纯化的角质形成细胞呈典型的上皮形态,第3代角质形成细胞增殖曲线为S形,对数增长期的平均倍增时间为36.9 h,角蛋白阳性率为98.9%。结论建立高纯度的人2型糖尿病皮肤角质形成细胞的无血清培养方法。

Objective To improve the methods for primary culture and purification of type 2 diabetic human kerati- nocytes in serum-free medium in vitro. Methods Type 2 diabetic human keratinocytes were isolated by 2 step di- gestion of neutral protease and trypsin, cultured in serum-free medium and purified by two-step subculture, the ke- ratinocytes were observed using inverted phase contrast microscope, the growth curve was obtained by the WST-8 assay and phenotype and purity were analyzed by flow cytometry. Results Type 2 diabetic human keratinocytes in serum-free medium showed typical epithelial morphology, the growth curve of the keratinocyte at passage 3 is S-shaped, the average doubling time is 36.9 hours during logarithmic growth phase and the keratin-positive rate was 98.9%. Conclusions The methods for culture of high-purity type 2 diabetic human keratinoeytes in serum-free media have been established.