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  

Different expression of oxidative stress and related pathways between human hypertrophic scar and normal skin fibroblasts

作　　者：李仕一 杨锦秀 赖琳英 周桂文 付强[1] 梁黎明[1] 陈敏亮[1] Li Shiyi;Yang Jinxiu;Lai Linying;Zhou Guiwen;Fu Qiang;Liang Liming;Chen Minliang(Department of Burns and Plastic Surgery,the Fourth Medical Center of Chinese PLA General Hospital,Beijing 100048,China;Graduate School of Chinese PLA General Hospital,Beijing 100039,China)

机构地区：[1]解放军总医院第四医学中心烧伤整形医学部,北京100048 [2]解放军总医院研究生院,北京100039

出　　处：《中华整形外科杂志》2020年第11期1270-1277,共8页Chinese Journal of Plastic Surgery

基　　金：国家自然科学基金面上项目(81772085)。

摘　　要：目的研究人增生性瘢痕与正常皮肤组织成纤维细胞中氧化应激程度及相关通路的表达是否存在差异。方法选取并收集2019年6月至2020年7月期间就诊于解放军总医院第四医学中心烧伤整形医学部接受手术切除治疗的8例增生性瘢痕患者的瘢痕组织及供皮区正常全层皮肤组织,其中男5例,女3例,年龄(35.0±11.7)岁。组织块法提取培养原代成纤维细胞,并传代培养,待细胞传代至第3代进行以下实验:(1)CCK-8法检测增生性瘢痕成纤维细胞(HSF)和正常皮肤成纤维细胞(NSF)的增殖能力;(2)流式细胞术检测HSF和NSF中活性氧含量;(3)比色法检测2种细胞内总超氧化物歧化酶(SOD)相对活力值;(4)RT-PCR检测2种细胞中NQO1基因表达量;(5)RT-PCR检测2种细胞中Nrf2基因表达量;(6)蛋白质印迹法检测2种细胞中Nrf2和Bcl2蛋白含量;(7)细胞免疫组织化学染色检测Nrf2在2种细胞中的表达分布情况。结果采用SPSS 25.0统计软件分析,行独立样本t检验,P<0.05表示差异有统计学意义。结果(1)与NSF比较,HSF的生长速度更快,2种细胞的细胞数量从第3天起差异有统计学意义(统计结果从3~7 d依次为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)HSF组中活性氧含量为75.39%±3.06%,明显高于NSF组的45.36%±3.66%,两者比较差异有统计学意义(t=-17.804,P<0.001);(3)HSF组SOD活力值为(50.76±0.52)U/ml,比NSF组的(42.76±1.35)U/ml升高,两者比较差异有统计学意义(t=15.674,P<0.001);(4)HSF组中NQO1 mRNA相对表达量为0.859±0.076,比NSF组的1.595±0.181降低,两者比较差异有统计学意义(t=3.763,P=0.020);(5)HSF组中Nrf2 mRNA相对表达量为0.590±0.055,相较于NSF组的1.595±0.146降低,两者比较差异有统计学意义(t=6.445,P=0.003);(6)HSF组的Nrf2蛋白相对含量为0.314±0.035,相较于NSF组的0.912±0.039明显降低,两者比较差异有统计学意义(t=22.554,P<0.001);HSF组的Bcl2蛋白相对含量为0.466±0.020,相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

关 键 词：增生性瘢痕 氧化应激 成纤维细胞 活性氧 细胞凋亡 

分 类 号：R622[医药卫生—整形外科]