雌激素对人表皮干细胞增殖与迁移影响的体外实验

EFFECTS OF ESTROGEN ON PROLIFERATION AND MIGRATION OF HUMAN EPIDERMAL STEM CELLS IN VITRO

目的表皮干细胞(epidermal stem cells,ESCs)在体内的微环境十分复杂,雌激素可能是主要的参与者。探讨雌激素对体外培养的hESCs增殖与迁移的影响。方法取自愿捐赠的正常人包皮标本,采用密度梯度离心法分离培养hESCs,并传至第2代。流式细胞仪检测第2代hESCs整合素β1、细胞角蛋白19(cytokeratin 19,CK19)、CK14和CK10抗原表达以鉴定hESCs。取第2代hESCs 2×106个分别用含0.1 nmol/L雌激素的ESCs专用培养基(A组)、含10 nmol/L雌激素受体阻断剂的ESCs专用培养基(B组)以及普通ESCs专用培养基(C组)进行培养;于培养后24 h刮擦生长融合成片的hESCs,制备宽度为100μm的体外单层hESCs损伤模型。于模型制备后24、48、72 h,倒置相差显微镜下观察各组表皮创面愈合情况(即hESCs迁移情况),并计算模型制备后72 h各组创面愈合率;于模型制备后24、48、72、96、120 h,采用MTT法检测hESCs分裂增殖活性。结果原代培养的hESCs贴壁呈单层卵圆形、集落样生长。流式细胞仪检测第2代hESCs的整合素β1、CK19、CK14呈阳性标记,阳性率分别为96.63%、95.47%、94.27%;CK10呈阴性标记,阳性率为1.32%。模型制备后24、48、72 h,各组均可见hESCs迁移越过创缘,其中A组越过创缘的细胞数多于B、C组,C组多于B组;模型制备后72 h,A、B、C组创面愈合率分别为69.00%±0.05%、35.00%±0.05%、48.00%±0.06%,组间比较差异均有统计学意义(P<0.05)。模型制备后24、48、72、96和120 h,A组hESCs分裂增殖活性高于B、C组,C组高于B组,差异均有统计学意义(P<0.01)。结论雌激素具有促进hESCs分裂增殖和迁移的作用,并藉此可能参与皮肤诸多的生物学作用。

Objective In vivo,the microenvironment of epidermal stem cells（ESCs） is complex,and estrogen might be involved in the micro environment.To investigate the biological effects of estrogen on the proliferation and migration of ESCs in vitro.Methods hESCs were isolated from normal human foreskin and cultured.The second generation of hESCs were identified with flow cytometry after being marked with integrin β1,cytokeratin 19（CK19）,CK14,and CK10 antigens.hESCs of 2 × 106 cell density were cultured with ESCs special medium supplemented with 0.1 nmol/L Diethylstilbestrol in group A（estrogen group）,with ESCs special medium supplemented with 10 nmol/L Raloxifene hydrochloride in group B（ER blocking agent group）,and with ESCs special medium in group C（control group）,respectively.The 100 μm ＂scratch＂ wounds were created by scraping confluent hESCs plated on Petri dishes with a sterile pipette tip in vitro.The migrating cells from the wound edge were quantified at 24,48,and 72 hours after incubation.The rates of wound healing were calculated by SigmaScan Pro 5.0 software at 72 hours.The proliferating effect of estrogen on hESCs was determined with MTT method at 24,48,72,96,and 120 hours.Results Cultured primary hESCs could adhere to the wall showing ovoid in shape and grew into colonies.Flow cytometry showed the positive results for integrin β1,CK19,and CK14（with positive rate of 96.63%,95.47%,and 94.27%,respectively） and the negative result for CK10（with positive rate of 1.32%）.In group A,the number of cells crossing the wound edge was more than those of group B and group C at 24,48,and 72 hours.The rates of wound healing were 69.00% ± 0.05% in group A,35.00% ± 0.05% in group B,and 48.00% ± 0.06% in group C at 72 hours,showing significant differences among groups（P 〈 0.05）.The proliferating speed of hESCs was significantly higher in group A than in groups B and C（P 〈 0.01）,and significantly higher in group C than in group B（P 〈 0.01） at 24,48,72,96,and 120 hours.Conclusion The estrogen can promote the proliferation and migration of hESCs in vitro.It may be involved in many biological activity of skin.