| 题名 | 两栖类动物皮肤源性改造肽的功能研究 |
| 作者 | 唐璟 |
| 学位类别 | 博士 |
| 答辩日期 | 2014-11 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 赖仞 |
| 关键词 | 创伤愈合 MAPKs通路 Smads通路 |
| 其他题名 | Functional analysis of modified peptides derived from amphibian skin |
| 中文摘要 | 皮肤最基本的功能是作为一个保护性屏障抵抗外界环境和防止水分过度丢失，损伤或疾病导致的大面积皮肤完整性缺损可能引起严重的残疾甚至是死亡。皮肤受到损伤后会即刻启动创伤修复的过程，经过炎症反应、增殖和重塑而使伤口愈合。然而，这个修复过程会受到很多因素的影响，如局部缺血、糖尿病、静脉瘀滞、压力应激等，而导致创伤愈合障碍，甚或发展为非愈合性皮肤溃疡。据统计，美国的难愈合性皮肤修复障碍患者已高达300万人到600万人，每年造成的医疗保健支出总额超过30亿美元；而随着全球糖尿病发病率的升高，非愈合性糖尿病足溃疡（糖尿病的最常见并发症之一）导致糖尿病患者死亡的风险高达15% ~ 25%。因此，开发出强效促伤口愈合的药物已成为目前需要迫切解决的问题。 两栖类动物生活环境复杂，皮肤裸露、光滑、潮湿，容易受到病原微生物侵袭、紫外线和天敌毒素等的损伤。为适应复杂的栖息地和生态环境，在长期的自然选择过程中，两栖类逐渐进化出了独特有效的皮肤防御修复系统。两栖类动物皮肤因含有抗菌肽、抗氧化肽、蛙啡肽、神经毒素等多种药用活性肽而成为了一个天然的药物资源库。 海蛙（Fejervarya cancrivora）属于无尾目（Anura），叉舌蛙科（Dicroglossidae），叉舌蛙亚科（Dicroglossinae），陆蛙属（Fejervarya），海蛙是唯一能耐受海洋环境并能在海水中生活的两栖类动物。本实验室首次通过分离纯化、克隆等方法从海蛙皮肤鉴定得到两条与淡水栖息的印度虎纹蛙皮肤抗菌肽（tigerinin）共享特征性结构的tigerinin-RC1和tigerinin-RC2多肽，并发现这两个抗菌肽对于金黄色葡萄球菌、白色念珠球菌、枯草芽孢杆菌和大肠杆菌具有强烈的抗菌活性。 为探究海蛙皮肤源性肽在皮肤创伤愈合中的作用，我们以tigerinin-RC1和tigerinin-RC2的结构为基础，设计合成了一系列改造肽，分别命名为tiger 1 ~ tiger 12和tiger 17，并对它们可能存在的生物学功能进行了初步研究。结果表明：所有改造肽均未检测到明显的抗菌活性和丝氨酸蛋白酶抑制剂活性；MTT细胞增殖实验发现，样品tiger 17能显著促进人永生化表皮角化细胞（HaCat）和人皮肤成纤维细胞（HSF）增殖；细胞划痕实验结果显示tiger 17能促进HaCat角化细胞迁移。这些结果表明tiger 17和其基础的天然肽tigerinin-RC1及tigerinin-RC2功能不同，未表现出抗菌活性而具有促细胞增殖及迁移的活性，这表明tiger 17可能具有促进皮肤创伤修复的作用。 小鼠全皮层缺损创伤愈合被用来评估tiger 17的体内促创伤修复作用，实验结果表明，tiger 17能显著促进小鼠皮肤创面的收缩、加速创伤的愈合。H&E染色的组织切片显示tiger 17有利于创面组织的上皮化重建，创伤早期的肉芽组织形成，而在创伤修复后期tiger 17表现出促进肉芽组织收缩的能力。 从细胞机制的角度探索发现，tiger 17能以浓度依赖方式显著促进HaCat角化细胞的增殖并促进HaCat细胞迁移，这可能是tiger 17能加速上皮化重建的重要原因；此外，tiger 17对HSF细胞的促增殖及促迁移活性能很好解释tiger 17样品在创伤修复早期促进肉芽组织形成的作用。Matrigel基质胶体外管腔形成实验和体内鸡胚绒毛尿囊膜血管形成实验表明tiger 17能增强HUVEC细胞的管形成活性，可能有利于新生血管的形成。这些结果从细胞行为的层面阐释了tiger 17促皮肤创伤愈合的可能细胞机制。 对于操控细胞行为可能涉及的细胞因子我们通过ELISA进行了检测。用不同浓度的tiger 17样品刺激小鼠巨噬细胞RAW264.7，并检测可能涉及tiger 17促创伤愈合作用的细胞因子，结果显示tiger 17能促进RAW264.7细胞分泌促炎症细胞因子IL-6和生长因子TGF-β1，而对IL-1β、TNF-α、EGF的分泌没有明显影响。新生皮肤组织的免疫组化分析从体内角度支持了体外细胞因子检测结果：局部应用tiger 17显著趋化了巨噬细胞募集至创面区域，上调创区TGF-β1的表达，并促进成纤维细胞分化为具有强烈收缩表型的肌成纤维细胞。 Western blot检测发现，tiger 17刺激细胞后能显著促进MAPKs通路中重要信号蛋白JNK、Erk的磷酸化，而当使用MAPKs通路抑制剂阻断后，tiger 17的刺激无法引起TGF-β1分泌的上调，提示我们tiger 17促进TGF-β1产生可能是通过激活MAPKs信号通路来实现的。对TGF-β下游经典Smads通路的免疫印迹分析表明，tiger 17刺激细胞18小时后能显著增强Smad2/3信号蛋白的磷酸化水平，呈良好的剂量依赖性；当使用TGF-β1中和抗体与tiger 17共同孵育细胞18小时，不能再检测到磷酸化的Smad2/3条带，提示tiger 17可能以TGF-β1依赖性方式间接激活下游的Smads通路而引发一系列的细胞效应事件。 由于tiger 17易于合成、储存、运输的特点以及对内源性TGF-β1的促表达作用，我们认为tiger 17有望开发为外用促创伤愈合的优秀候选药物。 |
| 英文摘要 | The primary function of the skin is to serve as a protective barrier against the environment and excessive water loss. Loss of the integrity of large portions of the skin as a result of injury or illness may lead to major disability or even death. Skin-wound healing starts immediately after injury and consists of three phases: inflammation, proliferation, and remodeling. However, wound healing would be affected by many factors, such as ischemia, diabetes mellitus, venous stasis and pressure, and lead to impaired wound healing, even non-healing skin ulcer. The prevalence of nonhealing wounds is estimated to be between 3 and 6 million in the United States, and nonhealing wounds result in enormous health care expenditures with the total cost being estimated at more than $3 billion per year. The prevalence of diabetes mellitus is increasing worldwide, and non-healing diabetic foot ulcers (DFUs), one of the most common complications of diabetes, lead to a high lifetime risk of 15% to 25% in diabetic patients. Therefore, it is urgently necessary to develop more effective wound-healing medicines. The living environment of amphibian is complicated, and amphibian skin is directly exposed in biological and non-biological environment. Because of its uncovered, smooth and wet, amphibian skin is easily injured by lots of factors such as microorganism infection, radiation and predation poison. In order to adapt the complex habitat and environment, amphibian gradually developed the unique and efficient cutaneous defence system. Amphibian skin is an abundant bioactive treasury owing to numerous pharmacological bio-peptides such as antimicrobial peptides, antioxidative peptides, dermorphin and neurontoxin. Fejervarya cancrivora is a kind of the crab-eating frog belonging to Anura, Dicroglossidae, Dicroglossinae, Fejervarya. Sea frog is one of only a handful of amphibians worldwide that tolerates salinewaters. In our research of bioactive peptides of sea frog secretions, two novel 12-residues antimicrobial peptides were purified from the skin secretion of the crab-eating frog, Fejervarya cancrivora. These two antimicrobial peptides, tigerinin-RC1 and -RC2, share significant structural similarity with tigerinins found in the skin of Indian frog, Hoplovatrachus tigerinus. And cDNAs encoding tigerinin-RC1 and -RC2 were also cloned from the skin cDNA library of F. cancrivora. Moreover, it was proved that both tigerinin-RC1 and -RC2 showed strong antimicrobial activities for Staphylococcus aureus, Candida albicans, Escherichia coli and Bacillus subtilis. To investigate the promoting wound-healing ability of the peptides originated from sea frog cautaneous secretions, some modified peptides were designed and synthesized based on the sequences of tigerinin-RC1 and -RC2, named tiger 1 ~ 12 and tiger 17 respectively, and then these modified peptides were further explored for their possible biological functions. It was shown that all modified peptides did not have antimicrobial activities and serine protease inhibitor activity. By MTT assay, it was confirmed that tiger 17 obviously promoted HaCat cellline and HSF cellline cells proliferation. In vitro cells scratched assay, it was found that tiger 17 promoted HaCat cellline cells migration. From our results, it was supported that tiger 17 was differed from the natural original peptides, tigerinin-RC1 and -RC2, in other words, tiger 17 had no antimicrobial activities but had abilities of promoting cells proliferation and migration, which indicated the effects of tiger 17 on wound-healing. A full-thickness incision model in mice was employed to assess effects of tiger 17 on cutaneous wound healing in vivo. It was clearly shown that tiger 17 promoted wound contraction and accelerated wound healing. From H&E staining sections analysis, it was revealed that tiger 17 accelerated the neo-epithelial tongue reconstruction, the formation of granulation tissue, while tiger 17 promoted the contraction of gr |
| 语种 | 中文 |
| 内容类型 | 学位论文 |
| 源URL | [http://159.226.149.26:8080/handle/152453/10133]  |
| 专题 | 昆明动物研究所_动物毒素室 |
| 推荐引用方式 GB/T 7714 | 唐璟. 两栖类动物皮肤源性改造肽的功能研究[D]. 北京. 中国科学院研究生院. 2014. |
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