瘦素对皮肤哀老氧化应激水平影响的实验研究

发布时间：2018-07-17 06:16

【摘要】：目的：建立接近人体皮肤老化过程的快速、简便、经济的实验动物模型,结合皮肤老化的氧化应激学说,验证瘦素(Leptin)对皮肤老化过程中活性氧(ROS)生成及抗氧化防御能力的影响及调控,判明瘦素在延缓皮肤衰老过程中可能发挥的作用及其机制,从而为瘦素在抗皮肤老化甚至再生受损皮肤的应用寻找实验及理论依据,为皮肤抗衰老再生医学研究提供新的思路。方法：1.模拟人体皮肤衰老的内外源诱发因素,以D-gal注射联合UV辐照的方式,设置单一D-gal注射组、单一UV辐照组及空白对照组,从组织形态学(组织HE染色、Masson三色染色、弹力纤维染色)改变及氧化应激分子指标和代谢产物检测等变化,分别进行比较分析,寻找经济的实验动物、有效的干预措施和合理的干预时间,构建快速、经济、实用、周期短、更接近人体皮肤老化进程和规律的动物模型。2.在构建的皮肤老化动物模型基础上,设置空白对照和试验对照,试验组分为低(5ug/g)、中(10ug/g)、高(20ug/g)三个剂量组给予瘦素腹腔内注射,6周后取材对皮肤组织形态学和组织匀浆氧化应激生化指标进行比较,研究瘦素调控氧化应激、延缓皮肤老化的作用及可能的最适剂量。3.将瘦素溶液配制为低(5μg/ml)、中(10μg/ml)、高(20μg/ml)浓度,对培养的HSFs进行预处理,之后行UV辐照,诱导细胞老化。检测HSFs细胞活力、SA-β-gal活性,氧化应激生化指标及Western-blot测定HSFs中p67phox,PKCε和p66Shc蛋白的表达。研究瘦素延缓老化、调控氧化应激的可能机制。结果：1.对CD-1小鼠以每日UV辐照能量611.28mJ/cm2，隔日一次,联合D-gal剂量1mg/g/D,每日颈背部皮下注射的联合措施,与其他干预组对比,干预后6周可见明显的皮肤衰老组织形态学变化,氧化应激水平也在6周达到相对稳定状态,较其他干预组时间缩短、诱发老化效果更明显。2.瘦素系统应用,可明显缓解D-gal联合UV诱发皮肤衰老的组织学变化。瘦素在皮肤局部发挥出抗氧化作用,保护抗氧化酶、减少脂质过氧化和胶原蛋白的降解。同时发现,瘦素对抗皮肤老化进程中的氧化应激反应有剂量选择性,本实验中,中等剂量的瘦素(10ug/g)对衰老皮肤抗氧化酶的保护、拮抗脂质过氧化和胶原蛋白降解,能发挥更好的作用(P0.05)。3.瘦素干预可以缓解UV对人成纤维细胞抗氧化酶活性的降低作用及对膜脂质和胶原蛋白的破坏作用,可有效降低UV辐照诱导的成纤维细胞p67phox, PKCε和p66Shc表达增加。Leptin可以调控UV诱导的人成纤维细胞的氧化应激状态,缓解甚至逆转过氧化损伤,发挥细胞抗衰老甚至一定程度上的细胞再生作用。结论：细胞因子Leptin是一种多靶器官、多效应的蛋白激素。本实验研究发现,在动物和细胞层面,瘦素均可以发挥出拮抗氧化的作用,延缓皮肤老化的组织学及细胞学改变。其机制可能与Leptin拮抗或清除活性氧自由基、降低氧化应激反应有关。
[Abstract]:Objective: to establish a rapid, simple and economical animal model of skin aging, combined with the oxidative stress theory of skin aging. To verify the effect and regulation of leptin on the production of reactive oxygen species (Ros) and anti-oxidative defense during skin aging, and to identify the role and mechanism of leptin in delaying skin aging. So it can provide experimental and theoretical basis for the application of leptin in the application of anti-aging and even regenerating damaged skin, and provide a new idea for the research of anti-aging and regenerative medicine of skin. Method 1: 1. The internal and external inducing factors of human skin senescence were simulated. With D-gal injection combined with UV irradiation, single D-gal injection group, single UV irradiation group and blank control group were divided into three groups: histomorphology (HE staining) and Masson trichrome staining. The changes of elastic fiber staining and the changes of oxidative stress molecular indexes and metabolites were compared and analyzed respectively to find economic experimental animals, effective intervention measures and reasonable intervention time to construct fast, economical, practical, and so on. Short cycle, closer to the human skin aging process and law of the animal model. 2. On the basis of the animal model of skin aging, blank control and experimental control were set up. Three dose groups of low (5ug/g), middle (10ug/g) and high (20ug/g) groups were given leptin intraperitoneal injection for 6 weeks to compare the skin histomorphology and biochemical indexes of oxidative stress of tissue homogenate, and to study the regulation of leptin on oxidative stress. Effect of delaying skin aging and possible optimal dose. 3. Leptin solution was prepared with low (5 渭 g/ml), medium (10 渭 g/ml) and high (20 渭 g/ml) concentrations. The cultured HSFs were pretreated with UV irradiation to induce cell aging. The activity of SA- 尾 -gal, the biochemical index of oxidative stress and the expression of p67phoxton-PKC 蔚 and p66Shc protein in HSFs were detected by Western-blot. To study the possible mechanism of leptin delaying aging and regulating oxidative stress. The result is 1: 1. CD-1 mice were treated with UV irradiation energy of 611.28mJ / cm ~ (-2) every other day, combined with D-gal dose of 1mg / g / D and subcutaneous injection of D-gal daily. Compared with other intervention groups, obvious changes of skin aging histomorphology were observed 6 weeks after intervention. The level of oxidative stress also reached a relatively stable state at 6 weeks, which was shorter than that of other intervention groups, and the effect of inducing aging was more obvious. 2. The application of leptin system can significantly alleviate the histological changes of skin senescence induced by D-gal combined with UV. Leptin exerts antioxidation effect, protects antioxidant enzymes, and reduces lipid peroxidation and collagen degradation. At the same time, it was found that leptin had dose-selective effects on oxidative stress during skin aging. In this experiment, moderate dose of leptin (10ug/g) protected aging skin from antioxidant enzymes and antagonized lipid peroxidation and collagen degradation. Can play a better role (P0.05). 3. Leptin intervention alleviated the decrease of antioxidant enzyme activity and the destruction of membrane lipid and collagen protein in human fibroblasts induced by UV. The expression of p67phox, PKC 蔚 and p66Shc in human fibroblasts induced by UV irradiation can be effectively reduced. Leptin can regulate the oxidative stress state of human fibroblasts induced by UV irradiation and even reverse the peroxidation injury. Give play to cell anti-aging and even to a certain extent of the role of cell regeneration. Conclusion: the cytokine leptin is a multi-target organ and multi-effect protein hormone. In this study, it was found that leptin could antagonize oxidation and delay the histological and cytological changes of aging skin at both animal and cell levels. The mechanism may be related to leptin antagonizing or scavenging reactive oxygen free radicals and reducing oxidative stress.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R339.38

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