摘要
目的探讨表皮细胞体外去分化模型的建立方法。方法人表皮角质形成细胞（HEK）体外培养至24代，细胞体积膨大，形态发生明显改变。碱性成纤维细胞生长因子（bFGF）诱导48h后，观察细胞贴壁培养4、12、24、48、72h时的生长及集落形成情况。以同期培养的表皮细胞作为阴性对照，采用免疫组织化学的方法检测实验组及对照组，B1整合素、CK19、CK14、CK10在表皮细胞中的表达差异；流式细胞仪测定细胞周期的改变。结果bFGF诱导培养48h后老化的表皮细胞周围分散出现幼稚型细胞，这些细胞体积小，形态均一，折光性强，核浆比大，并呈克隆样生长。免疫组织化学染色结果显示，bFGF诱导培养后，表皮细胞中CK19、CK14表达增强，而CK10表达减弱。流式结果显示：bFGF诱导培养后，细胞周期中G0／G1的细胞为53．08％，S期细胞为32．71％，G2期细胞为14．21％，而对照组中处于G0／G1、S期及G2期的细胞分别为63．9％、18．98％及17．12％。结论bFGF能够在体外诱导表皮细胞逆转分化形成幼稚型克隆形成细胞，具体机制需要进一步深入研究。
Objective To establish the dedifferentiated model of epidermal cells induced by basic fibroblast growth factor （bFGF） in vitro. Methods Human epidermal cells became inflated and obviously alterd in shape, which resembled differentiated epidermal cells in vivo after 24 passages. The proliferation and clonogenesis of epidermal cells generated were examined at adherent time of 4,12,24,48 and 72 h after incubation with bFGF for 48 h. For controls, epidermal cells were cultured simultaneously in the absence of bFGF treatment. The expression levels of β1 integrin, CK19, CK14 and CK10 were detected by using immunohistochemical staining. For cell cycle analysis, cells samples were collected and analyzed by flow cytometry after propidium iodide （PI） staining. Results After treatment with bFGF for 48 h, clusters of round-shaped cells were detectable around differentiated epidermal cells, and expanded progressively thereafter. These cells were smaller in shape and mononucleated cells with large nuclear-cytoplasmic ratio, which led to not only clonogenicity but also ability to reconstitute an epidermal ridge like structure. Immunohistochemical staining revealed that the expression levels of CK19 and CK14 were up-regulated,while those of CKIO significantly down-regulated after bFGF treatment. For cell cycle analysis, after incubation with bFGF,the percent of cells in G0/Gl phase,S phase and G2 phase was 53.08% ,32.71% and 14. 21% respectively,and that in controls was 63.9% ,18.98% and 17.12% respectively,which represented the phenotypic changes between keratinocytes and dedifferentiated epidermal stem cells. Conclusion bFGF can reverse the differentiation of keratinocytes and induce them to produce immature cells, which can proliferate to generate clones.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2008年第3期391-393,共3页
Chinese Journal of Experimental Surgery
基金
国家重点基础研究发展计划资助项目（2005CB522603）
