缺氧对人真皮成纤维细胞向肌成纤维细胞表型转化的影响及其机制  

Effects of hypoxia on the phenotype transformation of human dermal fibroblasts to myofibroblasts and the mechanism

作　　者：赵彬[1] 韩夫[1] 张伟[1] 王许杰[1] 张健[1] 杨芳芳[1] 石继红[1] 苏琳琳[1] 胡大海[1] Zhao Bin Han Fu Zhang Wei Wang Xufie Zhang Jian Yang Fangfang Shi Ji- hong Su Linlin Hu Dahai(Burn Center of PLA, Department of Burns and Cutaneous Surgery, Xijing Hospi- tal, the Fourth Military Medical University, Xi＇an 710032, China)

机构地区：[1]第四军医大学西京医院全军烧伤中心,烧伤与皮肤外科,西安710032

出　　处：《中华烧伤杂志》2017年第6期368-373,共6页Chinese Journal of Burns

摘　　要：目的探讨缺氧对人真皮Fh向肌Fh表型转化的影响及其机制。方法取对数生长期第3代健康成人真皮Fb，用含体积分数10％FBS的DMEM培养基培养，进行以下5项实验。（1）实验1、2、3均将所取细胞按随机数字表法分为常氧组、缺氧组，每组10皿。常氧组细胞于含体积分数21％氧气环境中培养，缺氧组细胞于含体积分数1％氧气环境中培养。培养0、48h，2组分别取5皿细胞，实验1用实时荧光定量RT—PCR法检测细胞中肌Fh标志物α平滑肌肌动蛋白（α—SMA）、Ⅰ型胶原和Ⅲ型胶原mRNA表达，实验2用蛋白质印迹法检测细胞中α-SMA、Ⅰ型胶原和Ⅲ型胶原蛋白表达，实验3用蛋白质印迹法检测细胞中NF-κB蛋白表达。（2）实验4将细胞按随机数字表法分为常氧组、缺氧组、缺氧＋吡咯烷二硫代氨基甲酸盐（PDTC）组，每组5皿。前2组细胞同实验1处理，缺氧＋PDTC组细胞同缺氧组细胞处理并在培养基中加入4mL终物质的量浓度为10μmol／LPDTC。培养48h，蛋白质印迹法检测各组细胞中NF—κB蛋白表达。（3）实验5将细胞按随机数字表法分为常氧组、缺氧组、缺氧＋PDTC组、常氧＋PDTC组，每组5皿。前3组细胞均同实验4处理，常氧＋PDTC组细胞同常氧组细胞处理并在培养基中加入4mL终物质的量浓度为10μmol／LPDTC。培养48h，蛋白质印迹法检测各组细胞中α—SMA、Ⅰ型胶原和Ⅲ型胶原蛋白表达。对数据行析因设计方差分析、单因素方差分析、LSD—t检验。结果（1）与常氧组相应时相点比较，缺氧组Fb培养0h时α-SMA、Ⅰ型胶原、Ⅲ型胶原mRNA和蛋白表达量以及NF-κB蛋白表达量均无明显变化（t值为-1．21～2．04，P值均大于0．05），培养48h时α—SMA、Ⅰ型胶原、Ⅲ型胶原mRNA和蛋白表达量以及NF—κB蛋白表达量均显著升高（t值为-12．57～-3．44，P值均小于0．01）。（2）培养48h，缺氧组Fh中NF—κB�Objective To investigate the effects of hypoxia on the phenotype transformation of hu- man dermal fibroblasts to myofibroblasts and the mechanism. Methods The third passage of healthy adult human dermal fibroblasts in logarithmic phase were cultured in DMEM medium containing 10% fetal bovine serum for the following five experiments. （ 1 ） In experiments 1, 2, and 3, cells were divided into normoxia group and hypoxia group according to the random number table, with 10 dishes in each group. Cells of nor moxia group were cultured in incubator containing 21% oxygen, while those of hypoxia group with 1% oxy gen. At post culture hour （PCH） 0 and 48, 5 dishes of cells were collected from each group, respectively. mRNA expressions of markers of myofibroblasts including alpha smooth muscle actin （α-SMA） , type Ⅰ collagen, and type Ⅱ collagen of cells were determined with real time fluorescent quantitative reverse transcription polymerase chain reaction in experiment 1. Protein expressions of α-SMA, type Ⅰ collagen, and type Ⅲ collagen of cells were determined with Western blotting in experiment 2. The protein expression of nuclear factor-kappa B （NF-κB） of cells was determined with Western blotting in experiment 3. （2） In experiment 4, cells were divided into normoxia group, hypoxia group, and hypoxia ＋ pyrrolidine dithiocarbamate （PDTC） group according to the random number table, with 5 dishes in each group. Cells in the former two groups were treated the same as those in experiment 1. Cells in hypoxia ＋ PDTC group were treated the same as those in hypoxia group plus adding 4 mL PDTC with a final molarity of 10 μmol/L in the culture medium. At PCH 48, the protein expression of NF-κB of ceils was determined with Western blotting. （3） In experiment 5, cells were divided into normoxia group, hypoxia group, hypoxia ＋ PDTC group, and normoxia ＋ PDTC group according to the random number table, with 5 dishes in each group. Cells in the former three groups were treated th

关 键 词：缺氧 成纤维细胞 NF—κB 肌成纤维细胞 表型转化 

分 类 号：R622[医药卫生—整形外科]