| 其他摘要 | 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 |
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