摘要
目的:模拟表皮干细胞周围生态微环境,研究表皮干细胞在此环境中的表型变化,表皮干细胞微环境在表皮干细胞"命运"决定过程中的作用。方法:分离、培养表皮干细胞,观察其形态,并进行免疫组织化学鉴定及免疫荧光检测。制备小鼠胚胎组织匀浆液,荧光活细胞示踪法和逆转录聚合酶链式反应检测胚胎组织提取液对表皮干细胞表型变化的影响。结果:原代分离培养的表皮基底层干细胞表达α6、β1整合素、K19、K14、p63、Nes-tin、PCNA为阳性,而K10不表达。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。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、K19、K10表达增强,而K14表达减弱。结论:Ⅳ型胶原黏附法分离的表皮干细胞具有干细胞的特点。α6整合素、CD71在表皮干细胞中分布的区域差异及CD71表达位点的空间变化可以作为区分表皮干细胞及其子代细胞标志之一。胚胎组织提取液对表皮干细胞的增殖分化具有促进作用。胚胎组织提取液中的某些因子可能抑制了K14表达的TA细胞的终末分化,甚至诱导其发生逆向分化,导致诱导后K14的总体表达水平下调。
AIM. To investigate the phenotypic changes and cell - fate determination of keratinocyte stem cells in vitro. METHODS： Improved collagen Ⅳ-coated adhesion method was used to isolate and culture the keratinocyte stem cells 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 keratinocyte Stem cells （KSCs） were detected by flow FACS analysis and RT - PCR assays. RESULTS ： Immunocyto-chemical analysis proved that KSCs and their subunits were positive for α6 and β1 integrin, keratin 19 and 14, p63, nestin and PCNA, yet negative for keratin 10 expressions. Double staining immunofluorescence demonstrated that markers such as ors integrin and CD71 was co-expressed in KSCs, and there were important regional differences in the distribution of α6 integrin and CD71 in KSCs and transit amplifying cells （TA cells）. Meanwhile, two-color flow cytometric analysis of α6 integrin and CD71 consistently revealed that after treated with an extract from mouse fetuses, the percent of α6^＋ CD71^＋ and CD71^＋ fraction increased and reached to 68.43% and 4. 51% of the total isolated cells, respectively. However, the percent of α6^＋ CD71^- fraction reduced from 22. 49% to 10. 92% determined by flow cytometry. Moreover, the expression levels of β1 integrin and some putative biological markers in human epidermal cells were analyses by RT - PCR after fetus extract treatment. The relative gene expressions of β1 integrin, K19 and K10 increased in KSCs, whereas the expression of K14 in keratinocyte stem cells was observed to be significantly suppressed when compared to the appropriate controls.CONCLUSION ： The keratinocyte stem cells isolated by collagen Ⅳ- coated adhesion method have some characteristics of adult stem cells and perhaps the potentiality to differentiate and cross - differentiate. The regional differences in the distri- bution of α6 integrin and CDT1 are one of the transition markers to discriminate the KSCs and TA cells. Furthermore, fetus extracts contribute to not only the proliferation and self - renewal of KSCs but also the dedifferentiation of TA cell subunits.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2008年第5期883-887,共5页
Chinese Journal of Pathophysiology
基金
国家重点基础研究发展计划资助项目(No.2005CB522603)
关键词
表皮干细胞
抗体
单克隆
胚胎组织提取液
Epidermal stem cells
Antibodies, monoclonal
Embryonic tissue extracts
