Hoechst 33342标记恒河猴皮肤干细胞向结膜上皮细胞分化

 目的]探讨皮肤干细胞诱导分化为结膜上皮细胞,为组织工程结膜探索一种新型的种子细胞,为严重眼表疾病的治疗奠定基础。方法]在体外培养纯化鉴定恒河猴皮肤干细胞,并用Hoechst 33342标记,标记后的皮肤干细胞与结膜上皮细胞进行Transwell非接触共培养10d,然后对诱导后的细胞用细胞免疫化学以及流式细胞术检测结膜上皮细胞的特异性表面标志,包括角蛋白4和粘蛋白4,同时检测细胞的Hoechst 33342表达情况。结果]纯化后的皮肤干细胞比例接近90%。Hoechst 33342标记后的皮肤干细胞胞核显示蓝色荧光。在与结膜上皮细胞共培养10d后,皮肤干细胞显示结膜上皮细胞的标志粘蛋白4和角蛋白4阳性,流式细胞术检测阳性细胞比例分别为49.9%和95.6%,同时细胞还显示胞核的Hoechst 33342表达。结论]灵长类动物恒河猴的皮肤干细胞,在体外的适宜条件下,可诱导分化为有结膜上皮细胞特征的细胞。

Rhesus Monkey Epidermal Stem Cells Labelled by Hoechst 33342 Transdifferentiate to Conjunctival Epidermal Stem Cells

Objective To transdifferentiate epidermal stem cells of rhesus monkey to conjunctival epithelial cells in vitro. To investigate a new kind of seeds of artificial conjunctive. Methods Epidermal stem cells of rhesus monkey were cultured and separated. The isolated monkey epidermal stem cells were labeled by Hoechst 33342,and then co-cultured with conjunctival epithelial cells of rhesus monkey for 10 days. The pre-and post-co-culture epidermal stem cells were characterized with markers of conjunctival epithelial cells,mucin 4 and keratin 4,by immunocytochemistry and flow cytometry. And the nucleus of stem cells were detected by Hoechst 33342. Results The isolated epidermal stem cells of rhesus monkey were near 90% . After being labeled by Hoechst 33342,the nucleus of stem cells appeared blue fluorescence. After co-cultured with conjunctival epidermal stem cells for 10 days,the epidermal stem cells were strongly positive for mucin 4 and keratin 4. and the positive cells were 49.9% and 95.6% by flow cytometry. At the same time the nuleus of cells expressed Hoechst. Conclusion In suitable condition,epidermal stem cells of rhesus monkey can transdifferentiate to conjunctival epithelial cells by co-culturing with conjunctival epithelial cells in vitro.