丹参酮ⅡA对瘢痕成纤维细胞超微结构和自由基代谢平衡影响的实验研究

Effect of STS on cell ultrastructure and free radical balance of hypertrophic scar fibroblast in vitro

目的:探究丹参酮ⅡA磺酸钠(STS)对人增生性瘢痕(HS)的成纤维细胞(Fb)超微结构及自由基代谢平衡的影响,从抗氧化途径寻找一种新的治疗HS的有效方法。方法:收集术中7例HS组织进行Fb体外培养,取第3～6代传代细胞进行实验观察,设置实验组和对照组,实验组设置不同浓度组STS(0.05,0.075,0.10,0.125,0.15,0.20mg/ml)干预培养,对照组加入等量不含STS的DMEM培养液。①干预培养48h后应用倒置相差显微镜和透射电镜观察细胞形态及超微结构改变;②分别于培养12h,24h,48h后,应用化学比色法检测细胞内丙二醛(MDA)含量、黄嘌呤氧化酶(XOD)活力、总超氧化物歧化酶(T-SOD)活力及谷胱甘肽过氧化物酶(GSH-Px)活力变化。结果:①透射电镜见实验组细胞内线粒体肿胀及空泡样变性,粗面内质网扩张、囊泡变及脱颗粒,胶原纤维束减少,细胞坏死等现象;②与对照组比较,实验组MDA含量及XOD活力明显降低(P<0.05或P<0.01),且存在浓度-时间效应。与对照组比较,实验组T-SOD和GSH-Px活力水平有不同程度升高,其升高程度受药物浓度和作用时间影响。与对照组比较,药物作用12h时,高浓度组(0.15mg/ml和0.20mg/ml)T-SOD活力水平升高明显(P<0.05);24h时,0.125～0.20mg/ml组显著升高(P<0.05或P<0.01);48h时,0.05mg/ml～0.10mg/ml组显著升高(P<0.05或P<0.01),0.15mg/ml和0.20mg/ml组显著降低(P<0.01)。与对照组比较,药物作用12h时,0.075～0.15mg/ml组GSH-Px活力水平明显升高(P<0.05或P<0.01),24h和48h时0.05mg/ml和0.075mg/ml组明显升高(P<0.01),48h时0.20mg/ml组GSH-Px活力水平明显降低(P<0.01)。结论:STS可通过降低自由基产生及增强抗氧化酶活性调节瘢痕成纤维细胞自由基代谢平衡和生物学功能。

Objective To study the effect of Sodium Tanshinone II -A Tulfona（STS）on Cell ultrastructure and free radical balance in fibroblasts （Fb）of human hypertrophic scar （HS）and to look for a new medicine of anti-oxidation for treatment of HS. Methods Fibroblasts culture were performed with HS tissue （n=7） from operation. The third to sixth passage cells were divided into two groups, experimental group and control group. In experimental group, Fb cultured for different length times with DMEM medium and different concentrations STS （0.05,0.075,0.10,0.125,0.15,0.2 mg/ml）. In control groups, there was only DMEM medium and no STS. ①After being cultured for 48h, cell morphology and ultrastructure were observed with inverted phase contrast microscope and transmission electron microscope （TEM）. ② After being cultured for 12h,24h,48h, the changes of malondialdehyde （MDA） content, and xanthine oxidase （XOD）,total superoxide dismutase （T-SQD）,glutathione peroxidase （GSH-Px） activity levels in Fb were detected by ultraviolet spectrophotometer. Results were analyzed with medical statistical method. ResultsSwelling and vacuolar degeneration of mitochondria, rough endoplasmic reticulum vesicles change degranulation, collagen fibers decreased and cell necrosis were observed in Fb by TEM.②Compared with the control group, MDA content and XOD activity levels of the experimental group were significantly lower （P〈0.05 or P〈0.01）, and there is concentration - time effect. Compared with the control group, TSOD and GSH-Px activity levels increased to varying degrees, and it is affectedby the drug concentration and action time. At 12h,T-SOD activity levels were increased in high concentration group （0.15mg/ml and 0.20mg/ml）（P〈0.05） ;when 24h, T-SOD activity levels were significantly increased in 0.125-0.20mg/ml group （P〈0.05 or P〈0.01）; and those were significantly higher in 0.05~0.10mg/ml group at 48h （P〈0.05 or P〈0.01）, butsignificantly lower in 0.15mg/ml and 0.20 mg/ml group （P〈0.01）, compared with the control group. At 12h, GSH-Px activity levels were significantly increased in 0.075N0.15mg/ml group （P〈0.05 or P〈0.01）, when 24h and 48h, those were significantly higher in 0.05mg/ml and 0.075mg/ml group （P〈0.01）, but significantly lower in 0.20mg/ml group at 48h （P〈 0.01）, compared with the control group. Conclusion STS can regulate the free radicals balance and biological functions of fibroblasts by reduce the production of free radicals and enhance the activities of antioxidant enzymes.