蜕皮甾酮体外促进人表皮干细胞增殖的实验研究

目的：观察蜕皮甾酮（EDS）对人表皮干细胞增殖的影响。方法：中性蛋白水解酶-Ⅳ型胶原黏附法分离正常人包皮的表皮干细胞,免疫细胞化学法检测第3代细胞β1整合素、角蛋白19（K19）、K14、K10抗原表达以鉴定表皮干细胞。分别用EDS终浓度为0（对照组）、10、20、40、80、160mg/L的Epilife培养基培养表皮干细胞,于培养1d、2d、3d、4d时用3-（4,5-二甲基-2-噻唑）-2,5二苯基溴化四唑（MTT）法于酶标仪上检测490nm处的吸光度值（A值）,判定EDS对表皮干细胞体外增殖的影响。4d时采用流式细胞术检测各组细胞的周期改变,计算增殖指数（PI）。结果：免疫细胞化学法检测提示β1整合素、K19、K14抗原明显阳性,K10抗原阴性。培养1d、2d时,20、40、80mg/L组吸光度高于对照组（P〈0.05）,10、160mg/L组吸光度与对照组比较差异无显著性（P〉0.05）;培养3d、4d时,各实验组吸光度均高于对照组（P〈0.05）;各时间点所检测的吸光度值均以80mg/L时最高。流式细胞技术检测实验组PI值分别为40.00%、40.41%、40.96%、43.76%、40.87%,均高于对照组35.53%;实验组中以80mg/L时最高。结论：体外培养条件下EDS能促进人表皮干细胞的增殖,该促进作用在80mg/L时最为显著。

Experimental study on the effects of ecdysterone in promoting proliferation of human epidermal stem cells in vitro

Abstract Objective： To investigate the effects of ecdysterone（EDS） on the proliferation of human epidermal stem cells （hESCs） cultured in vitro. Methods： The hESCs were isolated from human foreskin by the method of protease-collagen iV-coated adhesion. β1-Integrin and keratin 19,14,10 as revealed by the aid of immunoeytochemistry were used as markers to identify the cells of the third generation of hESCs, hESCs were then randomly divided into 6 groups ： the hESCs in 5 experiment groups were respectively cultured in the Epilife with 10, 20, 40, 80, 160 mg/L of EDS, and in control group they were cultured in the Epilife without EDS. On days 1, 2, 3 and 4 after the hESCs were cultured, the optical density was measured to evaluate the effect of EDS. The cell cycles were determined with flow cytometry to calculate the proliferation index （PI） on day 4. Results： Immunoeytoehemistry showed the hESCs were positive for 131 intergrin, keratin 19 and keratin 14, but negative for keratin 10. After the cells were cultured for 1 and 2 days, the optical density of the experiment groups with EDS 20, 40, 80 mg/L were higher than that of the control group （P〈0. 05）, and there was no significant difference between the experiment groups with EDS 10, 160mg/L compared to the control group（P〉0. 05）. After the cells were cultured for 3 and 4 days, the optical density of all the experiment groups was higher than that of the control group（P〈0.05）. The optical density in the 80 mg/L of EDS group was higher than that of the other groups from 1-4 days. Flow eytometry showed PI of experiment groups was 40.00%, 40. 41%, 40.96%, 43.76%, 40. 87% respectively, which were higher than that of control group （35.53%）. PI of 80 mg/L of EDS group was the highest. Conclusion： EDS can promote the proliferation of hESCs in vitro at the optimal concentration of 80 mg/L.