Different expression of oxidative stress and related pathways between human hypertrophic scar and normal skin fibroblasts
10.3760/cma.j.cn114453-20200728-00446
- VernacularTitle:氧化应激及相关通路在人增生性瘢痕与正常皮肤组织成纤维细胞中的表达差异
- Author:
Shiyi LI
1
;
Jinxiu YANG
;
Linying LAI
;
Guiwen ZHOU
;
Qiang FU
;
Liming LIANG
;
Minliang CHEN
Author Information
1. 解放军总医院第四医学中心烧伤整形医学部，北京 100048；解放军总医院研究生院，北京 100039
- Keywords:
Hypertrophic scar;
Oxidative stress;
Fibroblasts;
Reactive oxygen species;
Apoptosis
- From:
Chinese Journal of Plastic Surgery
2020;36(11):1270-1277
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the expression of oxidative stress and related pathway factors in fibroblasts from human hypertrophic scar and normal skin tissue.Methods:Select and collect the scar tissue and normal full-thickness skin tissue of the donor area from 8 hypertrophic scar patients who were treated with surgical resection at the Department of Burns and Plastic Surgery, the Fourth Medical Center of Chinese PLA General Hospital from June 2019 to July 2020, extract and culture primary fibroblasts by weaving method, subculture them, and perform the following experiments when the cells were subcultured to the third generation: (1)Detect the proliferation ability of hypertrophic scar fibroblasts(HSF)and normal skin fibroblasts(NSF)with CCK-8 method.(2)Detect the number of reactive oxygen species in HSF and NSF by flow cytometry.(3)Colorimetric method for detecting relative activity of superoxide dismutase(SOD)in two kinds of cells.(4)Detect the NQO1 gene expression in two kinds of cells with RT-PCR.(5)Detect the Nrf2 gene expression in two kinds of cells with RT-PCR.(6)Detects Nrf2 and Bcl2 protein content in two kinds of cells with Western blotting.(7)Detect the expression and distribution of Nrf2 in cells with cellular immunohistochemical staining. The results were analyzed by SPSS 25.0 statistical software, independent sample t test was performed, and P<0.05 was considered as the difference was statistically significant. Results:(1) Compared with NSF, the number of the two kinds of cells is statistically different from the third day (The statistical results from the third day to the seventh day were as follows: t=2.631, P=0.039; t=7.025, P<0.001; t=5.031, P<0.001; t=4.241, P=0.002; t=6.525, P=0.003). (2) The reactive oxygen species content in HSF was 75.39%±3.06%, which was significantly higher than 45.36%±3.66% in the NSF group ( t=-17.804, P<0.001). (3) The relative activity of SOD of the HSF group was (50.76±0.52) U/ml, which is higher than that of the NSF group (42.76±1.35) U/ml ( t=15.674, P<0.001). (4) The relative expression of NQO1 mRNA in HSF was 0.859±0.076, which was lower than that in the NSF group at 1.595±0.181 ( t=3.763, P=0.020). (5) The relative expression of Nrf2 mRNA in HSF was 0.590±0.055, which was lower than that in the NSF group at 1.595±0.146 ( t=6.445, P=0.003). (6) The relative expression of Nrf2 protein of HSF was 0.314±0.035, which was significantly lower than 0.912±0.039 in the NSF group ( t=22.554, P<0.001). The relative expression of Bcl2 protein of HSF was 0.466±0.020, which was significantly lower than 0.734±0.066 in the NSF group ( t=7.780, P<0.001). (7) Nrf2 protein express in NSF and HSF cells, and protein expression was found in both nucleus and cytoplasm. Conclusions:HSF has a high degree of oxidative stress, which may be due to some reasons that reduce the content of Nrf2, resultsing in a decrease in the expression of various antioxidant enzymes, and ultimately leading to the accumulation of reactive oxygen species. Compared with NSF, HSF has stronger proliferation and apoptosis abilities, and reactive oxygen species can cause cell apoptosis, indicating that oxidative stress may be one of the pathogenesis of hypertrophic scars.
