Chinese Journal of Tissue Engineering Research ›› 2016, Vol. 20 ›› Issue (45): 6807-6813.doi: 10.3969/j.issn.2095-4344.2016.45.018
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Li Bing1, Wang Jun-ai2
Revised:
2016-09-12
Online:
2016-11-04
Published:
2016-11-04
About author:
Li Bing, Master, Attending physician, Department of Dermatology, the 451st Hospital of Chinese PLA, Xi’an 710054, Shaanxi Province, China
Supported by:
the National Natural Science Foundation of China, No. 31370769
CLC Number:
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.
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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