两种栖动物皮肤中促修复肽的结构和功能分析
文献类型：学位论文
| 作者 | 刘涵 |
| 学位类别 | 博士 |
| 答辩日期 | 2013-11 |
| 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 | 华南雨蛙 无指盘臭蛙 伤口愈合 动物模型 功能 表皮生长因子EGF |
| 其他题名 | Structure and Function Analysis of Wound-healing Promoting Peptides from Two Amphibians |
| 学位专业 | 动物学 |
| 中文摘要 | 皮肤作为生物体抵御外界环境侵害的第一道防线，对生物的正常生存起着至关重要的作用，但同时皮肤也成为最容易受到损伤的器官，因此当受到外界损伤时机体对皮肤的自我修复过程就变得尤为重要。虽然说皮肤损伤修复的过程是一个极其自然而普遍的过程，正常情况下当生物体皮肤受到损伤时，均会启动一系列应对和级联反应以促进伤口愈合和皮肤的快速修复，但是也有很多因素会影响伤口愈合过程中的某一个或某几个阶段，从而造成组织修复障碍甚至久伤不愈。据统计，在美国，难愈性皮肤修复障碍影响了300到600万人，其中65岁以上的老年人占了85%。难愈性皮肤修复障碍每年带来的巨额医疗花费总计超过30亿美元。开发出更有效地促伤口愈合的药物成为亟待解决的问题。 两栖类皮肤作为一个多功能器官，对于其生存发挥着重要作用。它直接暴露在一些生物和非生物损伤下，如微生物感染、寄生虫、捕食者以及物理性伤害如辐射和机械损伤等。两栖类皮肤作为一个巨大的资源库，里面包括了多种有药用活性的多肽分子，如抗菌肽、抗氧化肽、缓激肽、神经毒素等。虽然两栖类皮肤裸露、脆弱易受到伤害，但对于其皮肤伤口愈合的能力却知之甚少。之前已有文献报道用两栖类皮肤粗样涂抹在小鼠创面上对创面愈合有良好的促进作用，但却从未从两栖动物中鉴定出具体发挥促愈合作用的多肽。 在对华南雨蛙的皮肤活性多肽的研究中，我们通过分离纯化的手段从其皮肤分泌液中分离得到6条同源性很高的小肽序列，分别命名为hylareleasin1-3和9-11。经过一系列活性检测后我们发现这几条小肽中的hylareleasin 3有明显促进人脐静脉内皮细胞(Human umbilical vein endothelial cells，HUVEC)细胞增殖的活性。而后，我们通过对这几条小肽设计简并引物，从华南雨蛙皮肤cDNA文库中筛选得到这几条小肽的cDNA全序列，同时还得到另外5条同源性很高的cDNA序列，我们将其命名为predicted hylareleasin4-8，这5条序列与前面得到的6条序列具有很高的同源性，通过BLAST比对证实是一个新的小肽家族。其中的predicted hylareleasin4和hylareleasin3的成熟肽序列一样具有明显的促HUVEC细胞增殖的活性，同时酶联免疫吸附实验(Enzyme-linked immunosorbent assay， ELISA)的结果显示这两条小肽还具有促进HUVEC细胞释放表皮生长因子(Epidermal growth factor，EGF)和血管内皮细胞生长因子(Vascular endothelial growth factor，VEGF)的活性，而众所周知EGF和VEGF是促进皮肤修复的关键生长因子。这个结果提示我们华南雨蛙皮肤分泌液中存在潜在的能促进皮肤修复的小肽分子。 在对无指盘臭蛙的研究中，我们从无指盘臭蛙的皮肤抗菌肽中鉴定出一条具有促进皮肤伤口愈合能力的多肽，我们将其命名为AH90，它由24个氨基酸构成，其氨基酸序列是ATAWDFGPHGLLPIRPIRIRPLCG，分子量为2.6 kDa。AH90在细胞划痕的初步筛选实验中促角化细胞的迁移能力明显好于其他被测试的小肽。除了体外实验，在小鼠全皮层缺损的动物模型中它和作为阳性对照的EGF比显示出相当的促皮肤修复能力。苏木精-伊红(hematoxylin-eosin，HE)染色组织切片的病理学分析显示给药组小鼠皮肤创面中上皮化重建程度明显好于对照、同时肉芽组织厚度明显小于对照，另外表皮层厚度也明显小于对照，说明给药组的表皮增生程度不如对照明显。免疫组化的结果显示给药组皮肤创面中α-平滑肌肌动蛋白(α-smooth muscle actin，α-SMA)的表达出现明显的上调，从而促进了成纤维细胞向肌纤维母细胞的转化，这可能是造成伤口收缩和肉芽组织收缩的主要原因。ELISA的结果显示AH90能以浓度依赖的方式促进小鼠巨噬细胞Raw264.7中转换生长因子(Transforming growth factor， TGF)-β1的释放。AH90对伤口愈合的调节作用与三条信号通路相关，他们分别是丝裂原活化蛋白激酶(Mitogen-activated protein kinases，MAPK)、核因子κB (Nuclear factor，NF)-κB和TGF-β/Smad信号通路。免疫印迹结果显示AH90能以时间和剂量依赖的方式显著激活MAPK通路中的c-jun氨基端激酶(c-Jun NH(2)-terminal kinase，JNK)通路。此外，AH90还能以时间和剂量依赖的方式磷酸化IκB-α蛋白，促进p65蛋白的转核，与启动子上的转录元件结合促进下游目的基因的转录。而抑制剂实验显示：MAPK通路中的JNK和NF-κB信号通路确实参与到了由AH90促进TGF-β1释放的过程中去。而对于TGF-β/Smad信号通路，免疫印迹(Western blot，WB)结果显示AH90不能直接激活TGF-β/Smad信号通路，而是通过TGF-β依赖性的途径激活TGF-β/Smad信号通路。另外，它还能够通过上调角化细胞表面α5，α6整合素受体的表达而促进其在胞外基质蛋白如纤维连接蛋白和层粘连蛋白上的细胞黏附作用。 到目前为止，已经有很多种药物被开发用来治疗伤口愈合，一些生长因子如EGF就曾经被用来外用治疗一些外周组织的损伤以及口服和直肠静脉给药用于治疗胃肠道损伤等。除了价格不菲的生产成本外，这些生长因子的储存和运输不便等都限制了其临床应用。因此，一些具有促进内源性伤口愈合成分（如EGF，TGF-β等）的小分子多肽可能具有良好的临床应用前景。考虑到其易于生产、储存和运输以及能促进内源性生长因子的产生，从华南雨蛙和无指盘臭蛙中发现的这几条小分子多肽有望成为新型的治疗伤口愈合的候选药物分子，并能为设计其他促伤口愈合的小分子药物提供参照的模板。另外，这两项研究能为揭示两栖动物出色的皮肤修复能力提供线索和佐证。 |
| 英文摘要 | As the first line in defending the environmental damages, skin plays an important role in the survival of organisms. Meanwhile, skin becomes the most fragile organ. So it is important for the skin to possess self-repairment ability when injuried. Although skin wound healing is such a natural process that organisms will elicit self-repairment when it is injuried by the environmental damages, it has been reported that many factors will affect one or more phases of the normal wound healing process thus resulting in the impairment of wound healing. It is estimated that non-healing wounds affect about 3 to 6 million people in the United States, with persons 65 years and older accounting for 85% of these events. Non-healing wounds result in enormous health care expenditures, with the total cost estimated at more than $3 billion per year. Developing more effective wound-healing drugs is therefore a necessity. Amphibian skin is a versatile organ and plays key roles for the survival. It is directly exposed to biological or non-biological injuries such as microorganism infection, parasitization, predation, and physical harm including radiation and aseptic wound. Amphibian skin is a repertoire of biomaterials with lots of reported pharmacological functions, including anti-infection, anti-parasitization, anti-predation or anti-oxidation. Although amphibian naked skins are fragile and easy to be injured, little is known about its skin wound-healing ability. Although the application of amphibian skin for treating wounds has shown good effects, no detailed effective wound-healing promoting peptide has ever been characterised from amphibians before. In the research for the bioactive peptides from Hyla simplex’s skin secretion, six peptides sharing high homology were purified from the frog skin secretions, which were named hylareleasin 1-3 and 9-11, respectively. After subjecting to a number of pharmacological tests, hylareleasin 3 was found to significantly promote Human Umbilical Vein Endothelial Cells (HUVEC) cell proliferation. According to their amino acid sequences, the cDNA sequences encoding their precursors were cloned from the frog skin library. Other five precursors encoding hylareleasins were also obtained by the cDNA screening, which were named predicted hylareleasin4-8, respectively. Total 11 hylareleasins identified from this frog share high homology and by BLAST search, these peptides do not show any sequence similarity with known sequences, indicating that they are a novel family of peptides. Among them, predicted hylareleasin4 has also promoted HUVEC cell proliferation just as hylareleasin3. Meanwhile, both peptides can promote epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) releasing significantly from HUVEC cells. As we know, EGF and VEGF play important role in the regulation of wound healing. Thus, it may indicates that there are potential wound-healing promoting peptides in the skin secretion of Hyla simplex. A potential wound-healing promoting peptide (AH90, ATAWDFGPHGLLPIRPIRIRPLCG) with molecular weight of only 2.6 KDa was identified from the frog skin of Odorrana grahami. In an in vitro wound scratch assay, AH90 showed an outstanding cell migration promoting ability among all the tested peptides. Besides in vitro study, it showed similarwound healing-promoting activity withEGF in a murine model of full thickness dermal wound in vivo. Histological analysis indicated that mice treated with AH90 displayed accelerated epidermal and dermal regeneration, granulation tissue formation and deformation, and thinner epidermal thickness. Immunohistochemical analysis showed that protein level of alpha smooth muscle actin (α-SMA), the marker of myofibroblast differentiation, was significantly increased by AH90 treatment in the wounds, which might be responsible for the obvious wound area contraction and granulation tissue contraction. Three pathways related with wound healing were regulated by AH |
| 语种 | 中文 |
| 公开日期 | 2013-12-31 |
| 源URL | [http://159.226.149.42:8088/handle/152453/7761]  |
| 专题 | 昆明动物研究所_动物毒素室 |
| 推荐引用方式 GB/T 7714 | 刘涵. 两种栖动物皮肤中促修复肽的结构和功能分析[D]. 北京. 中国科学院研究生院. 2013. |
入库方式： OAI收割
来源：昆明动物研究所
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