[1]孙晓艳,付小兵,伊海英,等.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[J].第三军医大学学报,2008,30(21):2003-2007.
 SUN Xiao-yan,FU Xiao-bing,YI Hai-ying,et al.bFGF-induced dedifferentiation of epidermal cells into epidermal progenitor stem cells[J].J Third Mil Med Univ,2008,30(21):2003-2007.
点击复制

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(/HTML )
分享到:

《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
30卷
期数:
2008年第21期
页码:
2003-2007
栏目:
论著
出版日期:
2008-11-15

文章信息/Info

Title:
bFGF-induced dedifferentiation of epidermal cells into epidermal progenitor stem cells
作者:
孙晓艳付小兵伊海英刘惠玲孙同柱
解放军总医院:基础医学研究所;第一附属医院全军创伤修复重点实验室
Author(s):
SUN Xiao-yan FU Xiao-bing YI Hai-ying LIU Hui-ling SUN Tong-zhu
Insititute of Basic Medical Science, Beijing 100853;Wound Healing and Cell Biology Laboratory, Burns Institute, First Affiliated Hospital, General Hospital of PLA, Beijing 100037, China
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MTTRT-PCR
Keywords:
epidermal cells dedifferentiation stem cells immunohistochemistry flow cytometry MTT RT-PCR
分类号:
R322.991; R329.2; R341
文献标志码:
A
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100 ng/ml的碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)诱导液分别培养6、12、24、48、72 h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100 ng/ml,诱导时间为36~48 h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、CK14  mRNA转录水平明显上调,而CK10 mRNA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。
Abstract:
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 β1  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 CK10-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 immature, stem-like cells, which can proliferate and be used in the wound repair and regeneration of skin tissues.

参考文献/References:

孙晓艳, 付小兵, 伊海英, 等. 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[J]. 第三军医大学学报, 2008, 30(21):2003-2007.

相似文献/References:

[1]邹丽琴,苏踊跃,孙荣距,等.大鼠骨髓间充质干细胞向表皮细胞分化的初步研究[J].第三军医大学学报,2006,28(06):584.
[2]陈鑫,张一鸣,石小花,等.缺氧微环境下微管相关蛋白4在人表皮细胞中的表达变化及对细胞迁移能力的影响[J].第三军医大学学报,2013,35(22):2426.
 Chen Xin,Zhang Yiming,Shi Xiaohua,et al.Expression of MAP4 in epidermal cells and its effect on cell migration under hypoxic microenvironment[J].J Third Mil Med Univ,2013,35(21):2426.
[3]李伟,伍津津,吴国选,等.人角质形成细胞库的建立[J].第三军医大学学报,2004,26(11):0.[doi:10.16016/j.1000-5404.2004.11.018 ]
 LI Wei,WU Jin jin,WU Guo xuan,et al.[J].J Third Mil Med Univ,2004,26(21):0.[doi:10.16016/j.1000-5404.2004.11.018 ]
[4]杜太超,赵雄飞,赵阳兵,等.核酶抑制表皮细胞HLA-Ⅰ类抗原表达的实验研究[J].第三军医大学学报,2000,22(03):0.[doi:10.16016/j.1000-5404.2000.03.015 ]
 DU Tai chao,ZHAO Xiong fei,ZHAO Yang bin,et al.[J].J Third Mil Med Univ,2000,22(21):0.[doi:10.16016/j.1000-5404.2000.03.015 ]
[5]杜太超,赵雄飞,路淑珍,等.一种稳定、有效的表皮细胞基因导入方法的确立[J].第三军医大学学报,1999,21(09):0.[doi:10.16016/j.1000-5404.1999.09.024 ]
[6]王旭,吴军,王甲汉,等.复合皮覆盖烧伤创面的实验研究[J].第三军医大学学报,1996,18(03):0.[doi:10.16016/j.1000-5404.1996.03.003 ]
 Wang Xu,Wu Jun,Wang Jiahan,et al.[J].J Third Mil Med Univ,1996,18(21):0.[doi:10.16016/j.1000-5404.1996.03.003 ]
[7]陈鸿书,彭家和.猪脾淋巴细胞抑素的提取及其作用机制的研究[J].第三军医大学学报,1979,01(03):0.[doi:10.16016/j.1000-5404.1979.03.006 ]
[8]周鑫,黄竞卓,陈鑫,等.Gαi1蛋白促进短期低氧条件下表皮细胞迁移的实验研究[J].第三军医大学学报,2019,41(04):296.
 ZHOU Xin,HUANG Jingzhuo,CHEN Xin,et al.Gαi1 promotes migration of epidermal cells exposed to shortterm hypoxia [J].J Third Mil Med Univ,2019,41(21):296.
[9]蔡飒,付小兵,雷永红,等.在体诱导表皮细胞去分化的初步实验研究[J].第三军医大学学报,2008,30(21):1998.
 CAI Sa,FU Xiao-bing,LEI Yong-hong,et al.Dedifferentiation of human epidermal cells in nude mice[J].J Third Mil Med Univ,2008,30(21):1998.
[10]张翠萍,陈鹏,付小兵,等.Wnt/β-catenin信号通路活化对人表皮细胞表型变化的影响[J].第三军医大学学报,2010,32(16):1716.
 Zhang Cuiping,Chen Peng,Fu Xiaobing,et al.Effect of Wnt/β-catenin pathway activation on phenotypic characteristics of epidermal cells in vitro[J].J Third Mil Med Univ,2010,32(21):1716.

更新日期/Last Update: 2008-10-20