Effect of adipose-derived mesenchymal stem cell exosomes on the proliferation and migration of keratinocytes and the underlying mechanisms

doi:10.3969/j.issn.2095-4344.1527

Abstract

Abstract:

BACKGROUND: Skin wounds caused by burns and scalds are diseases of high morbidity and mortality rate, and it is urgent to find an effective way to facilitate wound healing.
OBJECTIVE: To investigate whether adipose-derived mesenchymal stem cell exosomes can vary the proliferation and migration of keratinocytes and the potential mechanisms.
METHODS: Human keratinocytes were cultured using trypsin digestion method, and identified by detection of keratinocyte specific marker protein Keratin 14. Exosomes from adipose-derived mesenchymal stem cells were obtained by ultracentrifugation and identified by the exosome specific marker proteins CD9, CD63 and CD81. The purified exosomes were added to keratinocytes at different concentrations (0, 10, 20, 50, 100 mg/L) to detect the proliferation and migration of keratinocytes at different time points (0, 12, 24, 48 hours).
RESULTS AND CONCLUSION: Adipose-derived mesenchymal stem cell exosomes facilitated the proliferation and migration of keratinocytes. The greatest improvement in the proliferation of keratinocytes was detected when the mass concentration was 50 g/mL after 48 hours of treatment. Meanwhile, the phosphorylation of ERK, AKT and STAT3 was strengthened with the existence of exosomes, indicating that adipose-derived mesenchymal stem cell exosomes may promote the proliferation and migration of keratinocytes through promoting the phosphorylation of ERK, AKT and STAT3.

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: Keratinocytes, Adipose Tissue, Mesenchymal Stem Cells, Exosomes, Cell Proliferation, Cell Movement, Tissue Engineering

CLC Number:

R459.9

Tian Xinli, Jiang Bo, Yan Hong. Effect of adipose-derived mesenchymal stem cell exosomes on the proliferation and migration of keratinocytes and the underlying mechanisms[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(1): 68-73.

Figures/Tables 1

2.1 人ADSCs和人角质形成细胞的分离和鉴定结果分离出的ADSCs呈成纤维样形状，且增殖率高，见图1A。为了进一步确定分离细胞即为ADSCs，对细胞进行成脂诱导分化培养2周，油红O染色呈阳性，见图1B。第3代人角质形成细胞均分裂活跃，胞体饱满，个体较小呈骰子状，细胞形态均一性好，见图1C。进一步通过角质形成细胞特异标记蛋白Keratin14免疫荧光染色鉴定，提示所分离的细胞为角质形成细胞，见图1D。 2.2 人ADSCs来源外泌体的鉴定结果利用超速离心法分离ADSCs来源外泌体，通过免疫印迹法检测外泌体特异标记蛋白CD9、CD63和CD81表达，见图2。分离的外泌体表达CD9、CD63和CD81，而ADSCs并未或者很少表达外泌体特异标记蛋白，表明成功分离出ADSCs来源外泌体。 2.3 人ADSCs来源外泌体对角质形成细胞增殖的影响角质形成细胞分别加入不同质量浓度(10，20，50，100 mg/L) ADSCs来源外泌体培养48 h后，采用MTT法检测各组细胞的体外增殖情况，见图3A，在加入外泌体后，角质形成细胞的增殖速度显著高于PBS阴性对照组，且呈浓度依赖性增长，在质量浓度为50 mg/L时增殖最显著；随后加入50 mg/L ADSCs来源外泌体培养0，12，24，48 h，采用MTT法检测各组细胞的体外增殖情况，见图3B，外泌体能够显著促进角质形成细胞增殖，且随着时间的增加而增加。为进一步验证上述实验结果，角质形成细胞分别加入PBS和50 mg/L ADSCs来源外泌体培养48 h后，采用免疫印迹法检测细胞增殖标记物ki-67的表达水平，得到与MTT法相似的结果，见图3C和3D。以上结果提示，人ADSCs来源外泌体可以显著促进角质形成细胞在体外的增殖。 2.4 人ADSCs来源外泌体对角质形成细胞迁移的影响角质形成细胞加入50 mg/L ADSCs来源外泌体培养，利用细胞划痕法观察24 h和48 h后各组细胞的迁移情况，见图4，在加入外泌体后，角质形成细胞的迁移速度显著高于PBS阴性对照组，且随着时间的增加而增加。以上结果提示，外泌体可以显著促进角质形成细胞的迁移。 2.5 人ADSCs来源外泌体对角质形成细胞相关信号通路蛋白的影响研究发现，ERK1/2、AKT和STAT3信号通路在创面愈合过程中起到重要的作用，包括细胞增殖、迁移和血管生成等[26]。因此，实验检测ADSCs来源外泌体是否通过ERK1/2、AKT和STAT3信号通路进而影响其增殖与迁移，见图5。角质形成细胞加入50 mg/L ADSCs来源外泌体培养48 h后，ERK1/2、AKT和STAT3的磷酸化水平显著高于PBS阴性对照组。以上结果提示，外泌体可能通过ERK1/2、AKT和STAT3信号通路进而影响其增殖与迁移。"

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