Role of the ERK pathway in the proliferation and differentiation of human epidermal stem cells

doi:10.3969/j.issn.2095-4344.2016.45.018

Abstract

Abstract:

BACKGROUND: Epidermal stem cells (ESCs) cultured in vitro can easily become differentiated and aged, but have limited proliferation ability. These characters limit the application and development of ESCs in skin tissue engineering. Increasing understanding of the regulatory mechanism of ESCs proliferation and differentiation would lay the theoretical foundation for the clinical application of ESCs.
OBJECTIVE: To identify the role of the ERK pathway in the proliferation and differentiation of ESCs.
METHODS: ESCs were isolated and cultured in vitro. The activity of the ERK pathway was blocked by PD98059 and the activation of the ERK pathway was conducted by PMA or transfecting MEK1 recombinant plasmid to enable the overexpression of MEK1. The effect of activation or blockage of the ERK pathway on the proliferation and colony formation ability of ESCs was detected by MTT and colony formation assay, respectively. The expression of ESCs markers K19 and β1-integrin and differentiated cell marker K10 was detected by western blot.
RESULTS AND CONCLUSION: When the activity of the ERK pathway was blocked by PD98059, the proliferation and colony formation abilities of ESCs were inhibited and the differentiation was enhanced. On the contrary, activating the ERK pathway by PMA or upregulation of MEK1 enhanced the proliferation and colony formation abilities of ESCs but inhibited the cell differentiation. These results indicate that the ERK pathway plays an important role in the proliferation and differentiation of ESCs.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Stem Cells, Epidermis, Extracellular Signal-Regulated MAP Kinases, Cell Proliferation, Cell Differentiation, Tissue Engineering

CLC Number:

R394.2

Li Bing, Wang Jun-ai . Role of the ERK pathway in the proliferation and differentiation of human epidermal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(45): 6807-6813.

Figures/Tables 1

2.1 表皮干细胞标志蛋白的鉴定为了鉴定分离的细胞是否为表皮干细胞，使用Western blot方法鉴定表皮干细胞表面标志蛋白K19和β1-整合素，细胞分化标志蛋白K10的表达，结果表明分离的表皮干细胞表达K19和β1-整合素，表达极少量K10。培养7 d后表皮干细胞K19和β1-整合素表达减少，分化标志蛋白K10表达增加，说明在培养过程中表皮干细胞逐渐分化，见图1。 2.2 激活或抑制ERK通路对表皮干细胞增殖的影响为了研究ERK通路对细胞增殖的影响，使用ERK通路抑制剂PD98059抑制ERK通路的活性，使用ERK通路激活剂PMA或者过表达ERK通路上游激酶MEK1激活ERK通路，MTT法检测细胞增殖情况。结果发现，使用抑制剂PD98059抑制ERK通路后表皮干细胞增殖率下降，使用PMA或过表达MEK1激活ERK通路后细胞增殖率上升，表明ERK通路对表皮干细胞的增殖有促进作用，见图2。 2.3 激活或抑制ERK通路对表皮干细胞克隆形成的影响为了研究ERK通路对表皮干细胞克隆形成能力的影响，使用抑制剂PD98059抑制ERK通路，克隆形成实验检测表皮干细胞的克隆形成能力。结果发现使用抑制剂PD98059抑制ERK通路后表皮干细胞克隆形成数减少，使用PMA或过表达MEK1激活ERK通路后表皮干细胞克隆形成数增加，表明激活ERK通路可以增强表皮干细胞的克隆形成能力，见图3。 2.4 激活或抑制ERK通路对表皮干细胞分化的影响使用抑制剂PD98059抑制ERK通路的活性，使用PMA或者过表达MEK1激活ERK通路，继续培养7 d后检测表皮干细胞及分化细胞标志蛋白。结果发现，与对照组相比，抑制ERK通路后表皮干细胞标志蛋白K19和β1-整合素表达降低，分化细胞标志蛋白K10表达升高。激活ERK通路后表皮干细胞标志蛋白K19和β1-整合素表达升高，分化细胞标志蛋白K10表达降低，表明激活ERK通路可以抑制表皮干细胞的分化，见图4。"

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