碱性成纤维细胞生长因子诱导表皮细胞去分化的初步研究  

bFGF-induced dedifferentiation of epidermal cells into epidermal progenitor stem cells

作　　者：孙晓艳[1] 付小兵[1] 伊海英[2] 刘惠玲[1] 孙同柱[2] 

机构地区：[1]解放军总医院基础医学研究所,北京100853 [2]解放军总医院第一附属医院全军创伤修复重点实验室,北京100037

出　　处：《第三军医大学学报》2008年第21期2003-2007,共5页Journal of Third Military Medical University

基　　金：国家重点基础研究发展计划(973计划)(2005CB522603)~~

摘　　要：目的初步探索表皮细胞体外去分化模型的建立方法。方法HEKa细胞体外培养6～7代时开始出现老化迹象,细胞体积膨大,形态发生明显改变。加入浓度为100ng/ml的碱性成纤维细胞生长因子(basic fibroblast growth fac-tor,bFGF)诱导液分别培养6、12、24、48、72h后,MTT法检测bFGF对表皮细胞体外生长的促进作用。同时采用免疫细胞化学染色法、流式细胞检测技术、RT-PCR法检测bFGF诱导培养后,表皮细胞的表型及功能的改变。结果MTT检测结果显示浓度为100ng/ml,诱导时间为36～48h为诱导表皮细胞去分化的最佳实验条件。在bFGF的作用之下,老化的HEKa细胞周围开始出现新生的表皮干细胞样克隆。免疫细胞化学检测结果表明,实验组细胞中β1整合素、CK19、CK14的表达增强,而CK10作为终末分化细胞的标志,在bFGF诱导组中的表达明显降低。流式细胞检测分析显示bFGF作用后,实验组CK19表达阳性的细胞与对照组相比明显增多(分别为74.77%和15.74%);CK14表达阳性的细胞也由原来的67.26%增加至被检测细胞总数的87.14%。而bFGF诱导作用后,被检测细胞中表达CK10阳性的细胞数量较对照组明显下降(分别为4.56%和98.56%)。此外,RT-PCR检测结果同样再次支持了表皮细胞去分化的理论。在bFGF诱导组中,β1整合素、CK19、CK14mRNA转录水平明显上调,而CK10mRNA表达则明显下降。结论bFGF能够在体外诱导表皮细胞逆转分化形成幼稚型表皮前体干细胞,但其涉及具体机制需要进一步研究。Objective To investigate the dedifferentiation of epidermal cells into their progenitor stem cells induced by basic fibroblasts growth factors （bFGF） in vitro. Methods HEKa cells obtained from Cascade were found flattening and formation of cell-to-cell contacts after 6 to 7 passages, which resembled differentiated epidermal cells in vivo. To examine the effect of growth factors on the cell proliferative alterations, bFGF （ 100 ng/ml） was added into the culture medium for different periods （6, 12, 24, 48, or 72 h）, then the cell proliferation was measured by MTT assay. Phenotypic changes and the cell-fate determination of HEKa cells after bFGF treatment were detected by immunocytochemical assays, flow cytometry and RT-PCR analysis. HEK cells with no intervention treatment were used as a control. Results MTT assay proved that the optimal culture condition to induce the dedifferentiation of epidermal cells into their progenitors was to culture HEKa cells for 36 to 48 h when the addition of bFGF was 100 ng/ml. After treatment with bFGF for 48 h, clusters of round-shaped cells appeared around differentiated epidermal cells, and expanded progressively thereafter. These cells were smaller in shape and with larger nuclear/cytoplasm ratio, and had not only clonogenicity but also ability to form a cutaneous ridge-like structure. Immunohistochemical staining revealed that the expression levels of 131 integrin, CK19 and CK14 were up-regulated, while the expression of CK10 was significant down-regulated after bFGF treatment. Flow cytometry indicated that there were more CK19-positive and CK14-positive cells in the treatment group than in the control （74.77% vs 15.74%, and 87.14% vs 67.26% respectively）, but much lesser CKl0-positive cells （4.56% vs 98.56% ）. Additionally, the mRNA expression levels of β1 integrin, CK19 and CK14 were up-regulated after bFGF treatment, but that of CK10 was down-regulated. Conclusion bFGF can reverse the differentiated process of epidermal cells and induce them to produce im

关 键 词：表皮细胞 去分化 干细胞 免疫细胞化学 流式细胞检测 MTT RT—PCR 

分 类 号：R322.991[医药卫生—人体解剖和组织胚胎学] R329.2[医药卫生—基础医学]