【作者】 孙晓艳；

【导师】 付小兵；

【作者基本信息】 中国人民解放军军医进修学院 ， 创伤外科学， 2008， 博士

【摘要】 目的:从建立表皮细胞去分化模型入手,比较去分化来源的表皮干细胞和机体自身来源的表皮干细胞在功能和生物学特性等方面的异同点,为实现无延迟、无瘢痕完美修复及皮肤组织的再生提供新的实验参考。方法:采用免疫组织化学的方法探寻胎儿皮肤中的表皮干细胞（epidermal stemcells,ESCs）,研究不同发育时期ESCs在胎儿皮肤中分布与迁移。采用改良的Ⅳ型胶原铺板选择黏附法分离培养人ESCs,观察其形态及增值分化的特点,并进行细胞免疫组织化学鉴定。同时,提取小鼠胚胎发育中期组织液,模拟表皮干细胞壁龛微环境,采用免疫组织化学、免疫荧光染色、流式细胞检测及RT-PCR技术研究表皮干细胞在此环境中的表型变化,探讨表皮干细胞周围生长微环境在表皮干细胞“命运”决定过程中的作用。此外,本研究在前期的工作基础上,建立表皮细胞去分化研究的体内、体外实验模型,采用免疫组织化学、MTT法、免疫荧光染色、流式细胞技术、扫描及透射电镜检测技术、RT-PCR、Western-blot及TRAP法再次验证表皮细胞通过去分化途径形成机体内源性去分化来源的表皮干细胞,参与皮肤组织的创伤修复与再生。同时,从细胞表型、超微结构及生物学功能等方面比较去分化来源的表皮干细胞和机体自身来源的表皮干细胞二者之间的异同点。结果:表皮干细胞在从基底层经棘层和颗粒层到角质层的分化迁移过程中,经历了有序的成熟模式,这一过程与角蛋白的程序性表达密切相关;毛囊的发生、发育不仅与ESCs的增殖分化有关,同时受到毛乳头的诱导作用。改良的Ⅳ型胶原选择黏附法能够高效的分离表皮中的干细胞。这些细胞具有干细胞的形态特点表达α₆整合素,β₁整合素,CK19,CK14,p63,Nestin及PCNA为阳性,而CK10染色为阴性;激光共聚焦检测显示α₆整合素和CD71在表皮干细胞中的分布存在着区域性差异,可以作为区分标志之一,为ESCs及其亚群的分离与鉴定提供实验参考。此外,流式细胞检测显示胚胎组织提取液作用后α₆⁺CD71^-表达细胞由细胞总数的22.49%减少至10.92%,而α₆⁺CD71⁺、α₆^-CD71⁺表达细胞则分别由诱导前的62.29%及0.19%增加至诱导后的68.34%及4.51%。RT-PCR结果表明胚胎组织提取液作用后,表皮干细胞中β₁整合素、CKl9、CK10表达增强,而CK14表达减弱。本文在前期工作的基础上,建立了表皮细胞去分化研究的体内、体外实验模型,免疫组织化学染色结果显示去基底层的超薄皮片CK19和β₁整合素染色为阴性;而移植第3、5、7d后,存活皮片内CK19和β₁整合素染色阳性的细胞重新出现,并且呈多层分布;流式细胞仪检测结果表明移植前后α₆⁺CD71^-和α₆⁺CD71⁺细胞比例有明显差异。人细胞系HEKa体外培养6～7代时,开始出现老化迹象。细胞免疫组织化学检测结果显示此时细胞表达CK10为强阳性,而β₁整合素、CK19表达为阴性,CK14则呈现弱阳性染色。在浓度为100ng/ml的bFGF诱导培养36～48h后,老化的HEKa细胞之间开始出现新生的表皮干细胞样克隆。这些细胞体积较小,为圆梭形,形态均一、折光性强、胞质近中央处有圆形核,核浆比大,因此称之为去分化来源的表皮干细胞（dHEK细胞）。dHEK细胞不但具有克隆形成能力,而且具有强大的分裂增殖能力。随着培养的延长,dHEK细胞克隆的面积逐渐增大,相互连接成片,并且和周围的HEKa细胞之间相互嵌合形成表皮嵴样结构。免疫组织化学检测发现bFGF诱导培养后,实验组细胞中β₁整合素、CK19和CK14的表达增强;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CK14表达阳性的细胞与对照组相比明显增多（分别为87.14%和67.26%）。CK19表达阳性的细胞也由原来的15.74%增加至被检测细胞总数的74.77%。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和Western-blot检测结果同样再次支持了表皮细胞去分化的现象。在bFGF作用后,表皮细胞重新程序化,并获得其前体干细胞的某些潜在能力,表达表皮干细胞的一些未分化蛋白。另外,表皮干细胞和去分化来源的dHEK细胞之间有两个显著的相同点:（1）二者都表达表皮干细胞的表面标志,如β₁整合素、α₆整合素和CK19等;（2）二者都存在着α₆整合素和CD71表达的区域性分布差异。此外,表皮干细胞和dHEK细胞之间的差异点有:（1）CD71-PE和α6 integrin-FITC直标双色荧光流式检测分析显示表皮干细胞和去分化来源的dHEK细胞之间存在着细胞亚群的分布差异;（2）hTERT在表皮干细胞和去分化而来的干细胞中存在着亚细胞定位差异。结论:ESCs并非真正静止的未分化细胞,而是一种已经进入分化状态但是仍具有多向分化潜能的干细胞,其增殖分化行为不但受到自身程序化基因表达的影响,同时又受到其所处的微环境的调控。bFGF能够诱导表皮细胞在体外发生去分化,去分化而来的表皮干细胞与ESCs相比具有更加强大的分裂增殖能力,可以作为种子细胞应用于皮肤创伤修复与再生的相关研究之中。更多还原

【Abstract】 Objectives:To investigate the effect of bFGF on cell-fate determination of human epidermal cells and its signaling related mechanism in vitro,meanwhile make a comparison of the difference between epithelial stem cells and those derived from dedifferentiation,and try to search for a possibility for the dedifferentiated stem cells to be used in the wound repair and regeneration of skin tissues.Methods:The expression of cytokeratin10（CK10）,an established marker of the differentiated epidermal cells,integrinβ₁,cytokeratin19（CK19）,cytokeratin14（CK14）, and PCNA,which were expressed by epidermal stem cells and their subpopulations has been analysed in fetal skins during various stages of the development using immunohistochemical methods.Meanwhile,improved collagenⅣ-coated adhesion method was used to isolate and culture the epidermal stem cells（ESCs）after neutron-protease selectively digested the dermo-epidermal junctions.Morphological features and identification of these immature cells were studied by immunochemistry and confocal microscopy analysis.After treated with extracts from 14-day-old mouse fetuses at the concentration of 10%（w/v）,phenotypic changes and the cell-fate determination of ESCs were detected by flow FACS analysis and RT-PCR assays. Furthermore,human foreskin epidermal sheets were prepared as previously described, following a removal of basal stem cells using repeated collagenⅣadhesion methods. Then,the treated ultrathin epidermal sheet was transplanted into the fresh full-thickness skin wounds measuring about 1 cm in diameter on the back of athymic BALB/c nude mice.Grafted biopsies were removed at day 3,5 and 7 post-grafting for histological and immunohistochemical staining,and flow FACS analysis.Additionally,HEKa cell line was incubated with bFGF（100ng/ml）for 36 to 48hr,then MTT assay, immunochemistry and confocal microscopy analysis,flow FACS,electron microscopy, semiquantitative RT-PCR,western-blot and telomerase activity assays（TRAP）were used to detected the the phenotypic changes and cell-fate determination of epidermal cells after bFGF treatment,simultaneously,making a comparison of the difference between epithelial stem cells and those derived from dedifferentiation.HEK cells with no intervention treatment were used as a control.Results:Both integrinsβ₁ and K19 were coexpressed in the undifferentiated epidermis.A specific,developmentally downregulation ofβ₁ expression was observed in the basal layer at the period of follicular keratinization.Coinciding with the differentiation of the epidermis,differential expression of K14 was observed in the basal and spinous layers,while K10 was confined to the differentiated spinous and granular layers.At the period of interfollicular keratinization,the epidermal progenitor cells mainly localized in the basal layer of epidermis and outer sheath,bulge and hair matrix of follicles,which were positive for integrinβ₁,CK19,CK14 and PCNA.Secondly, immunocytochemical analysis proved that ESCs and their subunits were positive for a6 andβ₁ integrin,CK19 and 14,p63,Nestin and PCNA,yet negative for CK10 expression. Double staining immunofluorescence demonstrated that markers such asα₆ integrin and CD71 was co-expressed in ESCs,and there will be important regional differences in the distribution ofα₆ integrin and CD71 in ESCs and transit amplifying cells（TA cells）. Meanwhile,two-color flow cytometric analysis ofα₆ integrin and CD71 consistently revealed that after treated with an extract from mouse fetuses,the percent ofα₆⁺ CD71⁺ and CD71⁺ fraction increased and reached to 68.43%and 4.51%of the total isolated cells respectively.However,the percent of a₆⁺ CD71 fraction reduced from 22.49%to 10.92%determined by flow-cytometry.Moreover,the expression levels ofβ₁ integrin and some putative biological markers in human epidermal cells were analyses by RT-PCR detection after fetus extract treatment.The relative gene expressions ofβ₁ integrin,CK19 and CK10 increased in ESCs,whereas the expression of CK14 in keratinocytes stem cells was observed to be significantly suppressed when compared to the appropriate controls.After the establishment of the dedifferentiation model of human epidermal cells in vivo,morphological analysis using immunohistochemical and immunofluorescent staining indicated that the expression of CK14 and 19,p63,and PCNA was localized in spinous and granular layers at the early grafted stage,while CK10 antigen was detectable in middle and outer layers.With the elongation of grafted period,a developmentally specific up-regulation ofβ₁ integrin and CK19 was detected in the spinous and granular layers,when CK14,p63,and PCNA appeared extensively in the inner and middle layers of grafted biopsies with down-regulation of CK10 in the middle and outer layers.It was intrigue that some stem cell formed islands could be also seen in the spinous and granular layers between the basal layer and the stratum corneum, which were strongly positive forβ₁ integrin and cytokeratin 19.MTT-assay proved that the optimum cultural condition to induce the dedifferentiation of epidermal cells into their progenitors was to culture HEKa cells for 36 to 48 hr with the addition of bFGF at a dosage of 100ng/ml.After treatment with bFGF for 48hr,clusters of round-shaped cells became 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 ofβ₁ integrin,CK19 and CK14 were up-regulated,while the expression of CK10 was significant down-regulated after bFGF treatment.For flow FACS analysis,essentially all cells were strong CK14-staining compared with controls （87.14 and 67.26%,respectively）.Significantly,the fluorescence intensity of the CK19 population was upregulated from 15.74%to 74.77%,and which of the CK10 population decreased from 98.56%to 4.56%after bFGF treatment.Additionally,the expression levels ofβ₁ integrin and some putative biological markers in human epidermal stem cells were analyses by RT-PCR detection after bFGF treatment.The relative gene expressions ofβ₁ integrin,CK19 and CK14 increased in bFGF treatment group, whereas the expression of CK10 was observed to be significantly suppressed when compared to controls.Western-blot and TRAP also proved the dedifferentiated phenomenon of epidermal cells in our experimental system.When epithelial stem cells are compared with dedifferentiated stem cells treated with bFGF（dHEK cells）,two points seem noteworthy.First,in both cases the cells remain multipotent and express markers of keratinocyte stem cells.Second,in both cases there seems to exist the regional differences by detection ofα₆ integrin and CD71 double labeling.However, although there seemed no significant changes were observed regarding the ultra and three-dimensional structure of ESCs and dedifferentiated derived stem cells following analysis with electron microscopy,double-immunofluorescent staining showed that the percents of TA cell subpopulation in dedifferentiated epidermal stem cells treated with bFGF is much higher than that in ESCs.Importantly,there exists a differentance of subcellular localization and hTERT activity between ESCs and dedifferentiated derived stem cells.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,however,the mechanism involved in bFGF signaling on the induction of epidermal cells’ dedifferentiation is unclear,which needs further research.更多还原