转化生长因子β1对长波紫外线照射皮肤成纤维细胞线粒体DNA 4 977 bp缺失的影响

背景:长波紫外线与人体皮肤光老化关系密切,线粒体的损伤是细胞衰老和死亡的分子基础.目的:观察长波紫外线照射对体外培养的皮肤成纤维细胞的线粒体脱氧核糖核酸缺失损伤的影响,以及转化生长因子β1对长波紫外线引起的线粒体DNA缺失有无保护作用.为皮肤光老化研究提供实验依据.设计、时间及地点:实验于2007-03/2008-04于解放军第四军医大学西京医院全军整形外科研究所完成.材料:转化生长因子β1为PerProtech公司产品;线粒体DNA 4 977 bp引物由上海生工合成;长波紫外线光源为北京光学仪器厂生产:紫外线辐照计为北京师范大学光电仪器厂生产.方法:收集20-23岁成年男性包皮环切术后的皮肤组织12例,体外培养成纤维细胞.将细胞分组为对照组、长波紫外线累计照射达到30,60,90 J,cm~2组,半定量PCR检测DNA4 977 bp缺失情况.不同质量浓度转化生长因子β1(0.1,1,1 0μg/L)干预长波紫外线累积照射达90 J/cm~2的皮肤成纤维细胞.主要观察指标:观察不同累积照射剂量产生的DNA 4 977 bp缺失:观察累积照射剂量90 J/cm~2后不同浓度转化生长因子干预对DNA4 977 bp缺失的影响.结果:体外培养的皮肤成纤维细胞经长波紫外线照射,长波紫外线累积剂量为长波紫外线60 J/cm~2后发生线粒体DNA4977 bp缺失,长波紫外线90 J/cm~2时缺失加重.吸光度值和PCR产物电泳及条带密度扫描结果显示,在照射前2 h加入不同剂量转化生长因子处理后,大剂量组(10 μg/L)线粒体DNA表达降低,中、小剂量组与长波紫外线照射组比较,差异无显著性意义.结论:一定剂量(10μg/L)转化生长因子对体外培养的成纤维细胞线粒体DNA缺失起到保护作用.

Influence of transforming growth factor-beta 1 on mitochondrial deoxyribonucleic acid 4 977 bp deletion of human dermal fibroblasts

BACKGROUND: Ultraviolet light (UVA) has a close relationship with photoaging, and mitochondrial damage is a basis of coil senescence and death.OBJECTIVE: To explore the influence of UVA on the mitochondrial deoxyribonucleic acid of human skin fibroblasts, in addition, to discuss whether transforming growth factor β1 (TGF-β1) could relieve mitochondrial DNA (mtDNA) deletion. DESIGN, TIME AND SETTING: The experiment was performed at Department of Plastic Surgery, the First Affiliated Hospital of Fourth Military Medical University from March 2007 to April 2008.MATERIALS: TGF-β1 was purchased from PerProtech Company; rnitochondrial DNA 4 977 bp primer was synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd; UVA light was produced by Beijing Optical Instruments Factory; and the ultraviolet radiation meter was provided by Photoelectric Instrument Factory of Beijing Normal University.METHODS: Young adult's fibroblasts were obtained from 12 cases with posthectomy. Then the cells were divided into control,UVA irradiation (30, 60, 90 J/cm~2) groups. The mitochondrial DNA 4 977 bp deletion was detected by semi-quantitative PCR. After that, TGF-β1 with different doses (0.1, 1, 10 βg/L) were used to interfere the cells with UVA 90 J/cm~2 irradiation.MAIN OUTCOME MEASURES: DNA 4 977 bp deletion under different doses cumulative irradiation, as well as the effect of TGF-β1 on mtDNA 4 977bp deletion after irradiates UVA90 J/cm~2 were observed.RESULTS: Mitochondrial DNA 4 977 bp had deleted when irradiated with cumulative dose of 60 J/cm~2 UVA, the deletion was aggravated when the UVA dose arrived at 90 J/cm~2, The absorbance value, PCR electrophoresis and band scanning showed that the deletion of mitochondrial DNA 4 977 bp was reduced after adding TGF-β1 at 2 hours prior to irritation in the large dose (10 μg/L) group. However, the difference between the medium and small dose groups had no obviously significance.CONCLUSION: A certain dose of TGF-β1 (10 μg/L) has protective effect on mtDNA 4 977 bp deletion.