目的 观察表皮细胞去分化过程中人端粒酶反转录酶(hTERT)的表达差异及碱性成纤维细胞生长因子(bFGF)的影响.方法 采用bRGF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检测bFGF诱导培养后表皮细胞表型的改变,并用流式细胞法、免疫荧光染色及TRAP-银染法检测bFGF诱导培养后表皮细胞中hTERT的表达差异.以同等条件下未经bFGF处理的人表皮细胞为对照组,同期分离培养的人在体表皮干细胞为阳性对照组.结果 免疫细胞化学染色显示,bFGF诱导培养后表皮细胞中β1-integrin、CK19、CK14表达明显增强,而CK10表达明显降低.流式细胞仪检测显示,bFGF诱导后实验组、对照组及阳性对照组hTERT阳性的细胞亚群分别占被检测细胞总数的9841%、0.77%及99.76%.TRAP-银染法检测显示实验组hTERT表达活性明显上调,且与阳性对照组比较无显著差异.间接免疫荧光染色显示实验组去分化来源表皮干细胞和在体表皮干细胞中的hTERT分布存在着亚细胞位点差异.结论 bFGF可诱导表皮细胞去分化并重新获得表皮干细胞的表型,这一过程不但可引起hTERT的表达及活性改变,而且能从亚细胞水平上诱导其发生位点迁移.%Objective To investigate the expression of human telomerase reverse transcriptase (hTERT) in epidermal cells and the influence on basic fibroblasts growth factor (bFGF). Methods The precursor epithelial stem cells (ESCs) were derived by in vitro bFGF-induced dedifferentiation of human keratinocytes (HEK) cells. The phenotypic changes of derived ESCs were detected by immunocytochemical staining, and the hTERT expression was detected by flow cytometry, immunofluorescence staining and TRAP-silver staining. HEK without bFGF treatment were used as control, and ESCs simultaneously isolated from human epidermis were used as positive control. Results Immunohistochemical staining revealed that the expression of β1-integrin, CK19 and CK14 in derived ESCs was significantly up-regulated, while the CK10 expression was significantly down-regulated. Flow cytometry revealed that the percentage of hTERT+ cell subsets in experimental group (derived ESCs) , control group and positive control group was 98.41％, 0.77％ and 99.76％,respectively. TRAP-silver staining analysis indicated that the telomerase activity was significantly up-regulated in experimental group compared with that in control group, while there was no significantly difference between experimental group and positive control group.Furthermore, immunofluorescence assay revealed that there was difference in subcellular localization of hTERT between experimental group (derived ESCs) and positive control group (ESCs). Conclusion Basic fibroblast growth factor may induce epidermal cells to dedifferentiate and regain the phenotype of ESCs, which leads to the changes in hTERT expression and activity, and induces its subcellular shift of the locus.
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