Study on molecular mechanism of extracellular vesicles derived from adipose-derived stem cells regulating TGF-β-Smad signaling pathway to inhibit scar hyperplasia
10.3760/cma.j.cn114453-20200612-00361
- VernacularTitle:脂肪来源干细胞胞外囊泡调控转化生长因子β-Smad信号通路抑制瘢痕增生的分子机制研究
- Author:
Yuanzheng ZHU
1
;
Yangyan YI
;
Jiangwen WANG
;
Jiaying NIE
;
Zhaohui WANG
;
Shu WU
;
Juanmin YANG
Author Information
1. 南昌大学第二附属医院整形美容科 330006
- Keywords:
Scar hyperplasia;
Transforming growth factor;
Adipose-derived stem cells;
Extracellular vesicles;
Myofibroblasts
- From:
Chinese Journal of Plastic Surgery
2020;36(10):1114-1120
- CountryChina
- Language:Chinese
-
Abstract:
Objective:This study aims to explore the potential effects of adipose-derived stem cell-extracellular vesicles(ASC-EVs) on TGFβ-Smad signaling pathway during myofibroblast trans-differentiation in vitro. Methods:ASCs were isolated from liposuction and flow cytometry was used to detect the surface protein markers. ASC-EVs were isolated from the supernatant of the third to fifth generation ASCs, and the microscopic morphology was observed by transmission electron microscope. The particle size distribution was detected by nano-particle tracking analyzer NanoSight and the membrane surface marker proteins CD63, Alix and TSG101 were detected by flow cytometry. The uptake of EVs by dermal fibroblasts co-cultured with PKH67 fluorescence labeled ASC-EVs was observed by confocal microscope. Dermal fibroblasts were continuously induced by TGFβ1 for five days, and ASC-EVs at the dose of 50 and 100 μg/ml were added. The expression of α-SMA and Smad-2/3/4 were detected by immunofluorescence staining, RT-PCR and Western Blot.Results:The results of flow cytometry showed that the surface markers CD73, CD49d, CD90 and CD105 of the third generation ASCs, were positive, and CD34 and CD45 were negative. Under transmission electron microscope, ASC-EVs was a round membranous vesicle with clear edge and surrounded by bilayer phospholipid membrane. The particle size of more than 95% of the ASC-EVs was distributed between 30 nm to 261 nm, with an average of (166.0±86.1)nm. The specific marker proteins of extracellular vesicle, CD63, Alix and TSG101 were highly expressed. Under confocal microscope, ASC-EVs with green fluorescence were uptake by dermal fibroblasts and distributed in the cytoplasm, and part of ASC-EVs was distributed around the nucleus.TGF-β1 induced a significant increase in the expression of α-SMA in dermal fibroblasts, and the addition of 50 or 100 μg/ml ASC-EVs reduced the expression of α-SMA genes and proteins, but did not show a dose-dependent manner. The gene and protein expression changes of Smad-2/3/4 were consistent with α-SMA. In addition, ASC-EVs could significantly reduced the content of type Ⅰ collagen in the supernatant, but had no significant effect on the secretion of type Ⅲ collagen.Conclusions:The mechanism of ASC-EVs inhibiting scar hyperplasia may be closely related to the suppression of TGF-β-Smad signaling pathway in dermal fibroblasts, inhibition of myofibroblast trans-differentiation and reduction of type Ⅰ collagen secretion.
