bFGF诱导表皮细胞去分化形成短暂扩充细胞  

Dedifferentiation of epidermal cells into transit amplifying cells induced by bFGF

作　　者：孙晓艳[1] 刘惠玲[1] 付小兵[1] 

机构地区：[1]解放军总医院基础医学研究所,北京100853

出　　处：《南方医科大学学报》2010年第9期2041-2046,共6页Journal of Southern Medical University

基　　金：国家重点基础研究发展计划资助项目(2005CB522603);国家自然科学基金(30901564);北京市科技新星计划资助项目(2008B53)~~

摘　　要：目的探索一条非转基因的途径诱导表皮细胞去分化形成表皮前体干细胞。方法采用bFGF诱导表皮细胞去分化形成表皮前体干细胞,免疫细胞化学染色法、流式细胞检测技术、RT-PCR法以及免疫荧光染色技术检测bFGF诱导培养后表皮细胞的表型及功能改变。同期分离培养的人在体表皮干细胞作为本次实验的阳性对照。结果免疫细胞化学染色表明bFGF诱导培养后,实验组细胞中β1-integrin、CK19、CK14的表达增强,而CK10的表达明显降低。流式细胞技术检测显示bFGF诱导后实验组、对照组及阳性对照组中α6+CD71-、α6+CD71+及CD71表达阳性的细胞亚群分别占被检测细胞总数的13.24%、58.26%、23.12%,0.12%、3.06%、51.50%及37.49%、45.13%、5.86%。RT-PCR检测结果显示,实验组中β1-integrin、CK19、CK14的mRNA转录水平明显上调,而CK10的mRNA表达则明显下降。虽然β1-integrin、CK19和CK10的表达在实验组和阳性对照组中没有显著性的差异(P>0.05),但CK14在实验组中的表达则显著性增高达1.4倍左右(P<0.05)。此外,免疫荧光染色结果显示端粒酶逆转录酶在去分化来源表皮干细胞和在体表皮干细胞中存在着亚细胞位点分布差异。结论 bFGF能够诱导表皮细胞去分化并且从新获得干细胞的潜能。虽然这些去分化来源的表皮干细胞在形态和功能上更加接近短暂扩增细胞,但是该方法期待能为表皮细胞去分化理论的深入认识以及探询一条高效、安全的iPS细胞制备途径提供实验参考。Objective To explore the method for inducing the dedifferentiation of epidermal cells into their progenitor stem cells in vitro without external gene intervention.Methods HEK cells obtained from Casacade were induced to reverse their differentiated process and produce immature stem-like cells,namely the dedifferentiation derived epidermal stem cells（dESCs）,by induction with basic fibroblasts growth factors（bFGF） in vitro.Immunochemical staining,flow FACS analysis,RT-PCR and immunofluorescent staining were used to detect the phenotypic and functional changes of the differentiated epidermal cells,using human epidermal stem cells（ESCs） as the positive control.Results Immunohistochemical staining revealed that the expressions of β1-integrin,CK19 and CK14 were up-regulated,while CK10 expression was down-regulated significantly after bFGF treatment.Two-color flow cytometric analysis of α6-integrin and CD71 showed that the percentages of α6＋CD71-,α6＋ CD71＋ and CD71＋ expressing populations reached 13.24%,58.26%and 23.12%of the total isolated cells,as compared with those of the control（0.12%,3.06%,51.50%） and positive control cells（37.49%,45.13%,5.86%）.RT-PCR analysis indicated that the relative gene expressions of β1-integrin,CK19 and CK14 increased in bFGF treatment group,whereas the expression of CK10 was significantly suppressed.Although there was no significant difference in the expression levels of β1 integrin,CK19 and CK10 between the bFGF-treated and the positive controls,the expression of CK14 in bFGF-treated cells showed a 1.4-fold increase as compared with that in ESCs（P〈0.05）.Immunofluorescent staining showed that a regional difference in the subcellularlocalization of telomerase between dESCs and ESCs.Conclusion bFGF can induce the epidermal cells to convert into epidermal precursorcells.Although they are more likely to be transient amplifying cells,the method for reprogramming somatic epidermal cells into their progenitors by bFGF induction other than genetic manipulat

关 键 词：表皮干细胞 去分化 IPS细胞 免疫细胞化学 流式细胞检测 RT-PCR 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]