摘要： 目的： 分离、培养并鉴定SD大鼠表皮干细胞；观察骨桥蛋白重组因子(recombinant rat osteopontin, rrOPN)对表皮干细胞体外增殖作用及促IL-6和IL-1β炎性因子分泌作用的影响。方法： 体外分离培养大鼠表皮干细胞，用倒置显微镜及电镜观察其形态，采用β1-整合素、CK15、CK19、CK5及CK10染色鉴定,并进行细胞生长曲线测定,流式细胞仪分析及细胞克隆形成率测定,以角质形成细胞作为对照。实验对不同浓度rrOPN处理的表皮干细胞采用MTT法测定细胞生长率，ELISA法测定IL-6、IL-1炎性因子分泌的变化。结果： 倒置显微镜观察所得鼠表皮干细胞呈上皮样生长，电镜观察细胞胞核大，较多处于分裂象。免疫组织化学检测表皮干细胞β1-整合素、CK15、CK19、CK5及CK10染色均呈阳性。细胞周期分析表皮干细胞中48.9%的细胞处于DNA合成期,细胞克隆形成率为34.7%。5～50 ng/mL的rrOPN促进角质形成细胞生长，浓度为15 ng/mL的rrOPN促进细胞生长作用最显著(P<0.05)。rrOPN引起IL-6明显升高(P<0.05)，IL-1β未见明显上调。结论： 成功建立大鼠表皮干细胞分离、培养方法，rrOPN促进表皮干细胞增殖，并促其炎性因子分泌，影响创口愈合。

关键词: 表皮干细胞, 骨桥蛋白（OPN）, 细胞培养, 创口愈合, 鼠

Abstract: Objective: The present study was designed to investigate the effects of recombinant rat osteopontin (rrOPN) on the proliferative effects of epidermal stem cells in vitro and the secretion of IL-6 and IL-1β by the cells. Methods: The epidermal stem cells were isolated from SD rats and cultured in vitro. The cell growth was observed by an inverted microscope and electron microscope. Immunocytochemistry was used to observed the expression of β1-integrin, keratin 15(CK15), keratin 19 (CK19), keratin 5 (CK5) and keratin 10 (CK10). The characteristics of the growth curve were explored, and the cell cycle was determined with flow cytometry (FCM) analysis and the colony forming efficiency (CFE) was studied. Keratinocytes were served as controls. The epidermal stem cells were treated with rrOPN at different concentrations. The influences of rrOPN on the proliferation of epidermal stem cells were checked by MTT assay. The secretion levels of IL-6 and IL-1β in culture supernatant were determined with ELISA. Results: Epidermal stem cells showed an epithelial-like appearance under  inverted microscopy. Many mitotic figures were observed by electron microscopy. Positive expression of β1-integrin, CK15, CK19, CK5 and CK10 of cultured cells were detected by immunocytochemistry. The cell cycle profile analysis showed that 48.9% cells were in the S stage. The CFE of the epidermal stem cell was 34.7%. Concentration of rrOPN ranging from 5 ng/mL to 50 ng/mL could stimulate the proliferation of epidermal stem cells, while  most remarkable at the concentration of 15 ng/mL. The IL-1β levels were significantly increased after induced with rrOPN (P<0.05), but the IL-6 levels had no statistically changed (P＞0.05). Conclusion: Skin Epidermal stem cells of rats can be successfully isolated and cultured in vitro; and rrOPN stimulated the proliferation of epidermal stem cells, and the secretion of IL-1β in epidermal stem cells could be induced with rrOPN.

Key words:  epidermal stem cell, osteopontin(OPN), cell culture, would healing, rat

中图分类号: 

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• R329.28