二甲基乙二酰基甘氨酸对瘢痕疙瘩成纤维细胞的增殖和胶原合成的影响  

Effects of dimethyloxalyl glycine on proliferation and collagen synthesis of keloid fibroblasts

作　　者：白石[1] 吴文艺[1] 汪怡[1] 黄种心[2] 朱世泽[1] 吴友谊[1] 王朝阳[1] 周仙颖 

机构地区：[1]福建医科大学附属第二医院整形外科,泉州362000 [2]福建医科大学附属第二医院病理科,泉州362000

出　　处：《中华实验外科杂志》2016年第10期2330-2333,共4页Chinese Journal of Experimental Surgery

基　　金：泉州市科技计划项目（Z-2014-0156）

摘　　要：目的 观察二甲基乙二酰基甘氨酸（DMOG）对体外培养瘢痕疙瘩成纤维细胞的增殖、胶原合成的影响.方法 使用不同浓度DMOG分别作用于体外培养的瘢痕疙瘩成纤维细胞,用噻唑蓝（MTT）法检测其增殖水平的差异和药物半数抑制浓度（IC50）及最佳干预时间;并将瘢痕疙瘩成纤维细胞分为对照组、DMOG组（IC50为800 μmol/L的DMOG）、缺氧组及缺氧＋DMOG组（缺氧＋800μmol/L的DMOG）,作用36 h（最佳干预时间）后用反转录-聚合酶链反应（RT-PCR）法、Western blot检测各组的缺氧诱导因子-1α（HIF-1α）、Ⅰ、Ⅲ型胶原的mRNA和蛋白表达水平;MTT法检测各组细胞的增殖水平,流式细胞仪检测各组的细胞周期;羟脯氨酸比色法检测各组细胞的胶原合成.结果 随着浓度的增加（200、400、800、1 600 μmol/L）,DMOG对瘢痕疙瘩成纤维细胞的增殖抑制呈剂量时间依耐性;在瘢痕疙瘩成纤维细胞中,缺氧组、DMOG组和缺氧＋DMOG组的HIF-1α的蛋白（0.98±0.09、1.36±0.16、1.43±0.21）和mRNA（1.14 ±0.11、1.24±0.16、1.27±0.11）的表达均较对照组的蛋白（0.71 ±0.13）和mRNA（1.01 ±0.02）高（P＜0.01）.流式细胞结果显示缺氧组、DMOG组和缺氧＋ DMOG组的G0/G1期细胞比例[（56.7±2.2）％、（61.3±3.3）％、（73.4±2.5）％],较对照组[（48.8±2.4）％]明显增加（P＜0.01）,S期细胞和G2/M期细胞比例均减少（P＜0.01）.与对照组比较,其他3组细胞增殖均受到了抑制,并且其中DMOG组和缺氧＋DMOG组培养36 h后细胞吸光度较对照组明显降低（0.54±0.01、0.29±0.02比1.01 ±0.02,P＜0.01）;与对照组比较,缺氧组中的羟脯氨酸水平、Ⅰ、Ⅲ型胶原的mRNA和蛋白有均所升高（P＜0.01）.与对照组比较,DMOG组和缺氧＋ DMOG组中的羟脯氨酸（11.86 ±0.64、14.72±0.03比15.92±0.22）、Ⅰ、Ⅲ型胶原的mRNA（0.63 ±0.07、0.86±0.08、0.56 ±0.09、0.82 ±0.10比1.00±0.01、1.00 ±0.03）和蛋Objective To investigate the effects of dimethyloxaloyl glycine （DMOG） on the proliferation and collagen synthesis of cultured human keloid fibroblasts in vitro.Methods in vitro cultured keloid fibroblasts were treated with different concentrations of DMOG.The methyl thiazol tetrazolium （MTT） assay was used to detect the difference in their proliferation levels and the median inhibitory concentration （IC50） and the best intervention time.Keloid fibroblasts were divided into control group and DMOG group （IC50 =800 μmol/L for DMOG）,hypoxia group and hypoxia ＋ DMOG group （hypoxia ＋800 μmol/L DMOG）.After 36 h （best intervention time）,reverse transcriptase-polymerase chain reaction （RT-PCR） and Western blotting were used to detect hypoxia inducible factor （HIF）-1ot,and mRNA and protein expression levels of type Ⅰ collagen A1 （COL1A1） and type Ⅲ collagen A1 （COL3A1）.MTT assay was used to detect the cell proliferation,and cell cycle was detected by flow cytometry.Collagen synthesis was measured by hydroxyproline colorimetric method.Results With the increases in the concentrations of DMOG （200,400,800,and 1 600 μmol/L）,DMOG inhibited the proliferation of fibroblasts in the keloid fibroblasts in a time-and concentration-dependent manner.The expression levels of HIF-1α protein （0.98 ±0.09,1.36 ±0.16,1.43 ±0.21） and mRNA （1.14 ±0.11,1.24 ±0.16,1.27 ±0.11） in hypoxia group,DMOG group and hypoxia ＋ DMOG group were higher than those in control group （0.71 ±0.13 and 1.01 ±0.02,P 〈0.01）.Flow cytometry showed that the proportion of G0/G1 phase cells in hypoxia group,DMOG group and hypoxia ＋ DMOG group [（56.7 ± 2.2） %,（61.3 ±3.3）%,and （73.4 ±2.5）%] was significantly increased as compared with that in control group [（48.8 ±2.4）%] （P 〈 0.01）,and the proportion of S phase cells and G2/M phase cells decreased （P 〈0.01）.As compared with the control group,the cell proliferation in other three groups were inhibited,and the ab

关 键 词：二甲基乙二酰基甘氨酸 瘢痕疙瘩 缺氧诱导因子-1Α 增殖 胶原 

分 类 号：R622[医药卫生—整形外科]