鼠表皮干细胞的分离培养及鉴定.docVIP

  鼠表皮干细胞的分离培养及鉴定# 李琴，王佐林** （同济大学附属口腔医院口腔种植科，上海 200072） 5 10 15 20 25 30 35 40 摘要：目的：分离、培养及鉴定 SD 大鼠表皮基底层干细胞。方法：中性蛋白水解酶选择性 的消化表皮与真皮之间的细胞连接，分离培养大鼠表皮基底层干细胞，倒置显微镜及电镜观 察，β1－整合素、CK15、CK19、CK5 及 CK10 染色鉴定，细胞生长曲线测定，细胞克隆形 成率测定，及流式细胞仪分析，以角质形成细胞作为对照组。结果：倒置显微镜观察所得鼠 表皮细胞呈上皮样生长，电镜观察细胞胞核大，较多处于分裂相，大量的粗面内质网。原代 分离的表皮基底层干细胞 β1－整合素、CK15、CK19、CK5 及 CK10 表达呈强阳性。细胞周 期分析表皮干细胞 48.9%细胞处于 DNA 合成期。细胞克隆形成率为 34.7%，明显高于表皮 角质形成细胞。结论：成功建立了大鼠表皮干细胞分离、培养的方法。 关键词：表皮干细胞；细胞培养；鉴定；创口愈合 Isolation, culture and identification of epithellial stem cells of rat Lin Qin, Wang Zuolin (Department of Oral Implantology, Hospital of Stomatolgy, Tongji University, Shanghai 200072) Abstract: Objective: To explore a method for isolating, culturing and identifying epidermal stem cells of rat. Methods: The epidermal stem cells were isolated and cultured after neutronprotease selectively digested the dermo-epidermal junctions. With observation of the cell growth by 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 was 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. Results: With inverted microscope, the cells were epithelial-like. With electron microscope, many mitotic figures can be observed. Immunohistochemical staining revealed that these cells were positive for β1-integrin, CK15, CK19, CK5 and CK10. 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%. Conclusion: Skin Epidermal stem cells of rats were successfully isolated and cultured in vitro. Key words: Epithelial stem cells； Cell culture; Identification; Wound healing 0 引言 皮肤是再生能力较强的组织,皮肤外层的表皮终身不断自我更新,而表皮干细胞的无限增 殖和分化,是表皮组织结构更新的基础。毛囊具有周期性生长的特性,能通过自身的干细胞进 行自我更新,现在多数研究认为毛囊干细胞位于毛囊的隆突部(bulge)和外根鞘的最