培养基血清浓度对人表皮干细胞增殖分化的影响doc.docVIP

培养基血清浓度对人表皮干细胞增殖分化的影响 南京大学医学院附属鼓楼医院 李云剑 林 樾 燕 辛 周宏礽 谭 谦 【摘要】目的:比较不同血清浓度培养体系对表皮干细胞增殖分化的影响。方法: 采用两步酶消化法和Ⅳ型胶原差速贴壁相结合的方法获得人原代表皮干细胞,分别以0％、5％、10％、15％和20％血清浓度的培养基在96孔板中进行培养。观察表皮干细胞形态，克隆形成及增殖的情况，应用四甲基偶氮唑蓝 MTT 比色法检测各组细胞存活和生长情况,分析量效和时效关系；持续传代培养细胞，每次传代的同时取适量细胞，用免疫细胞化学的方法进行表皮干细胞和表皮细胞相应标志物（K19、K14和K10）的测定。结果：表皮干细胞在各种血清浓度的培养基内均能形成克隆，增殖良好。用四甲基偶氮唑蓝 MTT 比色法测定，所得相同时间点各组OD值在统计学上没有差异（P＞0.05），表皮干细胞生长速度各组间无差异。第1代表皮干细胞K19均有表达，而K14和K10表达均为阴性；其后高血清浓度（15％、20％）培养基中细胞较低血清浓度（0％、5％）先出现K14、K10蛋白的表达；培养至第10代是各组细胞均出现K10高表达，而K19、K14表达阴性。结论:在低血清浓度 0％、5％ 的培养基中表皮干细胞生长良好，且能够相对较好保持表皮干细胞的特性。 【关键词】 表皮干细胞 血清浓度 增殖分化 培养 【Abstract】 Objective To study the proliferation and differentiation of epidermal stem cells in medium with different level of serum. Methods to obtain the primary epidermal stem cell with two step enzyme digestion method and type IV collagen coated chosen methods. Culture the cell in medium with different serum level--0％、5％、10％、15％ and 20％. The shape and clone formation and proliferation of the cell was observed through inverted microscope, tested by the MTT METHOD Immunocytochemistry was used to observe the expression of corresponding marker such as K19、K14 and K10 on epidermal stem cells in every generation after passaging. Results the epidermal stem cell can form clones in every kind of serum-medium, with well proliferation. There was no statistically significant difference in the OD values between different time points using MTT test. The expressing of the K19、K14、K10 in levelled serum-medium did not have obvious difference at first, but there can detect gradual difference between these subgroups with different level of serum during passaging. Conclusion the epidermal stem cell will keep its characteristics and grow better in medium with lower serum. 【Keyword】epidermal stem cells level of serum proliferation and differentiation culture 近年研究表明，表皮干细胞 epidermal stem cell, ESC 是皮肤发生、修复和重建的关键“种子细胞”。建立体外表皮干细胞的分离、纯化和培养体系,探讨ESC增殖、分化和迁移等机制已成为皮肤创面修复领域的研究重点。其中，分离、诱导和调控ESC，是组织工程化皮