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表皮生长因子(EGF)在HaCaT细胞中的过表达促进脂肪来源干细胞(ADSCs)的增殖、迁移、侵袭以及向表皮干细胞免疫表型的转分化。

The Overexpression of Epidermal Growth Factor (EGF) in HaCaT Cells Promotes the Proliferation, Migration, Invasion and Transdifferentiation to Epidermal Stem Cell Immunophenotyping of Adipose-Derived Stem Cells (ADSCs).

作者信息

Mao Yueping, Ma Jianchi, Xia Yue, Xie Xiaoyuan

机构信息

Department of Dermatology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China.

Department of Dermatology, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, China.

出版信息

Int J Stem Cells. 2020 Mar 30;13(1):93-103. doi: 10.15283/ijsc18146.

Abstract

BACKGROUND AND OBJECTIVES

The application of adipose derived stem cells (ADSCs) in skin repair has attracted much attention nowadays. Epidermal growth factor (EGF) participates in the progress of skin proliferation, differentiation and so forth. We aimed to explore the role of EGF in the proliferation, invasion, migration and transdifferentiation into epidermal cell phenotypes of ADSCs.

METHODS AND RESULTS

ADSCs were extracted from adipose tissues from patient. Immunophenotyping was determined by flow cytometry. Overexpressed EGF or siEGF was transfected by lentiviruses. EGF was determined by enzyme linked immunosorbent assay (ELISA) or western blot. ADSCs and HaCaT cells were co-cultured by Transwell chambers. Conditioned medium (CM) was obtained from cultured HaCaT cells and used for the culturing of ADSCs. Cell viability was tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Invasion rate was measured by Transwell invasion assay and migration rate by wound healing test. mRNA and protein levels were measured by qPCR and western blot respectively. The extracted cells from adipose tissues were identified as ADSCs by morphology and immunophenotyping. The expression of EGF was up or down regulated constantly in HaCaT cell line after transfection. EGF overexpression upregulated the proliferation, migration and invasion rates of ADSCs, and EGF expression regulated the expression of cytokeratin-19 (CK19) and integrin- as well.

CONCLUSIONS

EGF could be served as a stimulus to promote the proliferation, migration, and invasion as well as the transdifferentiation into epidermal stem cell immunophenotyping of ADSCs. The results showed that EGF had a promising effect on the repair of skin wound.

摘要

背景与目的

脂肪来源干细胞(ADSCs)在皮肤修复中的应用如今备受关注。表皮生长因子(EGF)参与皮肤增殖、分化等过程。我们旨在探讨EGF在ADSCs增殖、侵袭、迁移以及向表皮细胞表型转分化中的作用。

方法与结果

从患者脂肪组织中提取ADSCs。通过流式细胞术进行免疫表型分析。用慢病毒转染过表达的EGF或siEGF。通过酶联免疫吸附测定(ELISA)或蛋白质印迹法检测EGF。通过Transwell小室将ADSCs与HaCaT细胞共培养。从培养的HaCaT细胞中获得条件培养基(CM)并用于培养ADSCs。通过3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐(MTT)法检测细胞活力。通过Transwell侵袭试验测量侵袭率,通过伤口愈合试验测量迁移率。分别通过qPCR和蛋白质印迹法测量mRNA和蛋白质水平。从脂肪组织中提取的细胞通过形态学和免疫表型分析鉴定为ADSCs。转染后,HaCaT细胞系中EGF的表达持续上调或下调。EGF过表达上调了ADSCs的增殖、迁移和侵袭率,并且EGF表达也调节了细胞角蛋白-19(CK19)和整合素-α的表达。

结论

EGF可作为一种刺激因子,促进ADSCs的增殖、迁移、侵袭以及向表皮干细胞免疫表型的转分化。结果表明,EGF在皮肤伤口修复方面具有良好的效果。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b53c/7119215/287a0f66a5a8/IJSC-13-093-f1.jpg

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