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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。
Aim The microenvironment of epidermal stem cells (ESCs) in vivo is complex, and estrogen may be the main participant. To investigate the effect of estrogen on the proliferation and migration of hESCs cultured in vitro. Methods From the normal donated foreskin specimens, the hESCs were isolated and cultured by density gradient centrifugation and transferred to the second passage. Flow cytometry was used to detect the expression of integrin β1, cytokeratin 19 (CK19), CK14 and CK10 antigens in the second generation hESCs to identify hESCs. 2 × 106 second generation hESCs were cultured in ESCs containing 0.1 nmol / L estrogen (group A), ESCs containing 10 nmol / L estrogen receptor blockers (group B) and Cultured ESCs-specific culture medium (Group C); hESCs grown and fused together were scratched 24 h after culture to prepare an in vitro monolayer hESCs injury model with a width of 100 μm. At 24, 48, and 72 h after model preparation, wound healing (ie migration of hESCs) in each group was observed under an inverted phase contrast microscope. Wound healing rates were calculated at 72 h after model preparation. 72,96,120 h, MTT assay hESCs proliferation activity. Results Primary culture hESCs adherent monolayer oval-shaped, colony-like growth. The second generation of hESCs were detected by flow cytometry. The positive rates of integrin β1, CK19 and CK14 were 96.63%, 95.47% and 94.27%, respectively. The positive rate of CK10 was 1.32%. 24 h, 48 h, 72 h after model preparation, hESCs migrated across the wound margin in each group, of which the number of cells crossing the wound margin in group A was more than those in group B and C, and more in group C than in group B. At 72 h after model preparation, The wound healing rates in group B and group C were 69.00% ± 0.05%, 35.00% ± 0.05% and 48.00% ± 0.06%, respectively. There was significant difference between the two groups (P <0.05). At 24, 48, 72, 96 and 120 h after model preparation, hESCs proliferative activity in group A was higher than that in group B and C, and in group C was higher than that in group B, with statistical significance (P <0.01). Conclusion Estrogen can promote the proliferation and migration of hESCs, and may take part in many biological effects of the skin.