新型自组装短肽对皮肤创伤快速修复过程研究
发布时间：2019-03-01 21:28
【摘要】：目的:采用新型自组装短肽R2I4R2和K2I4K2应用于细胞三维培养和皮肤创伤快速修复,并初步探索其作用机制。方法:刚果红染色和原子力显微镜(AFM)检测短肽R2I4R2和K2I4K2的宏观结构;圆二色光谱(CD)检测理化条件对短肽R2I4R2和K2I4K2二级结构的影响;红细胞裂解试验检测两条短肽对细胞膜的裂解作用;CCK-8和AO/EB染色检测短肽R2I4R2和K2I4K2对人皮肤成纤维细胞HFF-1在三维环境中增殖情况;流式细胞周期分析HFF-1细胞在短肽形成的三维体系中的周期变化;建立SD大鼠皮肤创伤修复模型,考察两条短肽对皮肤创伤修复的影响;HE染色和免疫组化检测短肽R2I4R2和K2I4K2对皮肤创伤修复过程的病理变化;Real-time PCR检测皮肤修复相关蛋白基因和通路相关基因表达量的变化;以Taq DNA聚合酶作蛋白稳定假设模型,通过紫外可见光谱和real-time PCR考察短肽R2I4R2和K2I4K2对蛋白分子的保护作用。结果:CD检测显示两条短肽都具有介于α-螺旋和无规卷曲之间的二级结构,且在不同的离子和温度条件下都能保持较为稳定的结构。刚果红染色和(AFM)结果表明两条短肽都能自组装形成纳米纤维支架,进而形成水凝胶。而红细胞裂解试验和CCK-8检测结果显示两条短肽均无细胞毒性,能够很好的应用于细胞三维培养和创伤修复。动物实验表明短肽R2I4R2使创伤修复速度提高约28%,而K2I4K2提高约34%。而HE染色结果显示皮肤伤口加入短肽后3-5 d炎症细胞减少,7 d有血管形成,15 d后肉芽组织纤维化,进入组织重塑期。免疫组化结果进一步明确在加入短肽R2I4R2和K2I4K2后,5 d时血管内皮细胞因子CD34减少,7 d时VEGF含量增多。Real-time PCR结果显示HFF-1细胞在短肽形成的三维体系中,PEGRF基因表达量降低,VEGF、Integrin-α3、Integrin-α5和β-tublin基因表达量都有明显升高。UV检测结果显示,短肽R2I4R2和K2I4K2使对假设模型分子Taq酶在高温下的半衰期提高了1.44和1.68倍。Real-time PCR结果表明短肽R2I4R2和K2I4K2使Taq酶的扩增效率提高了约1.75和1.86倍。结论:本研究采用的新型自组装短肽R2I4R2和K2I4K2二级结构稳定,都能应用于细胞的三维培养。且两条短肽都能刺激创伤处血管再生、促进成纤维细胞迁移而使皮肤创伤快速修复的作用。本研究将激发更多的短肽设计理念的开发和短肽在组织修复再生、稳定蛋白质等领域中的应用。
[Abstract]:Aim: to use a novel self-assembled peptide R2I4R2 and K2I4K2 in three-dimensional cell culture and rapid skin wound repair, and to explore the mechanism of its action. Methods: Congo red staining and atomic force microscope (AFM) (AFM) were used to detect the macrostructure of short peptide R2I4R2 and K2I4K2, and the effect of physical and chemical conditions on the secondary structure of R2I4R2 and K2I4K2 was detected by circular dichroism (CD). The lytic effect of two short peptides on cell membrane was detected by erythrocyte lysis assay, and the proliferation of HFF-1 by R2I4R2 and K2I4K2 on human skin fibroblasts in three-dimensional environment was detected by CCK-8 and AO/EB staining. Flow cytometry was used to analyze the cycle changes of HFF-1 cells in the three-dimensional system of short peptide formation, to establish a model of skin wound repair in SD rats, and to investigate the effects of two peptides on skin wound repair. HE staining and immunohistochemistry were used to detect the pathological changes of short peptide R2I4R2 and K2I4K2 in the process of skin wound repair, and Real-time PCR was used to detect the expression of skin repair-related protein genes and pathway-related genes. Using Taq DNA polymerase as the hypothetical model of protein stability, the protective effects of R2I4R2 and K2I4K2 on protein molecules were investigated by UV-vis spectroscopy and real-time PCR. Results: CD analysis showed that the two peptides had a secondary structure between 伪-helix and random curl, and could maintain a stable structure under different ion and temperature conditions. Congo red staining and (AFM) showed that both peptides were self-assembled to form nanofiber scaffolds and then hydrogels were formed. The results of erythrocytic lysis test and CCK-8 assay showed that the two peptides were non-cytotoxic and could be used in three-dimensional cell culture and wound repair. Animal experiments showed that short peptide R2I4R2 increased wound healing rate by about 28%, while K2I4K2 increased by about 34%. The results of HE staining showed that the number of inflammatory cells decreased at 3 d, angiogenesis occurred at 7 d, and the granulation tissue was fibrotic at 15 d, which entered the stage of tissue remodeling at 3 d and 5 d after the skin wound was added with short peptide. The results of immunohistochemistry further confirmed that after the addition of short peptide R2I4R2 and K2I4K2, the CD34 of vascular endothelial cell decreased on the 5th day, and the content of VEGF increased on the 7th day. Real-time PCR results showed that HFF-1 cells were in the three-dimensional system of short peptide formation. The expression of PEGRF gene was decreased, and the expression of VEGF,Integrin- 伪 3, Integrin- 伪 5 and 尾-tublin gene were significantly increased. Short peptides R2I4R2 and K2I4K2 increased the half-life of Taq enzyme at high temperature by 1.44 and 1.68 times respectively. The results of Real-time PCR showed that R2I4R2 and K2I4K2 increased the amplification efficiency of Taq enzyme by about 1.75 and 1.86 times. Conclusion: the novel self-assembled peptide R2I4R2 and K2I4K2 are stable in secondary structure and can be used in three-dimensional cell culture. Both peptides can stimulate the regeneration of blood vessels and promote the migration of fibroblasts to repair skin trauma. This study will stimulate the development of short peptide design concept and the application of short peptide in tissue repair and regeneration, protein stabilization and so on.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R641
本文编号：2432818
[Abstract]:Aim: to use a novel self-assembled peptide R2I4R2 and K2I4K2 in three-dimensional cell culture and rapid skin wound repair, and to explore the mechanism of its action. Methods: Congo red staining and atomic force microscope (AFM) (AFM) were used to detect the macrostructure of short peptide R2I4R2 and K2I4K2, and the effect of physical and chemical conditions on the secondary structure of R2I4R2 and K2I4K2 was detected by circular dichroism (CD). The lytic effect of two short peptides on cell membrane was detected by erythrocyte lysis assay, and the proliferation of HFF-1 by R2I4R2 and K2I4K2 on human skin fibroblasts in three-dimensional environment was detected by CCK-8 and AO/EB staining. Flow cytometry was used to analyze the cycle changes of HFF-1 cells in the three-dimensional system of short peptide formation, to establish a model of skin wound repair in SD rats, and to investigate the effects of two peptides on skin wound repair. HE staining and immunohistochemistry were used to detect the pathological changes of short peptide R2I4R2 and K2I4K2 in the process of skin wound repair, and Real-time PCR was used to detect the expression of skin repair-related protein genes and pathway-related genes. Using Taq DNA polymerase as the hypothetical model of protein stability, the protective effects of R2I4R2 and K2I4K2 on protein molecules were investigated by UV-vis spectroscopy and real-time PCR. Results: CD analysis showed that the two peptides had a secondary structure between 伪-helix and random curl, and could maintain a stable structure under different ion and temperature conditions. Congo red staining and (AFM) showed that both peptides were self-assembled to form nanofiber scaffolds and then hydrogels were formed. The results of erythrocytic lysis test and CCK-8 assay showed that the two peptides were non-cytotoxic and could be used in three-dimensional cell culture and wound repair. Animal experiments showed that short peptide R2I4R2 increased wound healing rate by about 28%, while K2I4K2 increased by about 34%. The results of HE staining showed that the number of inflammatory cells decreased at 3 d, angiogenesis occurred at 7 d, and the granulation tissue was fibrotic at 15 d, which entered the stage of tissue remodeling at 3 d and 5 d after the skin wound was added with short peptide. The results of immunohistochemistry further confirmed that after the addition of short peptide R2I4R2 and K2I4K2, the CD34 of vascular endothelial cell decreased on the 5th day, and the content of VEGF increased on the 7th day. Real-time PCR results showed that HFF-1 cells were in the three-dimensional system of short peptide formation. The expression of PEGRF gene was decreased, and the expression of VEGF,Integrin- 伪 3, Integrin- 伪 5 and 尾-tublin gene were significantly increased. Short peptides R2I4R2 and K2I4K2 increased the half-life of Taq enzyme at high temperature by 1.44 and 1.68 times respectively. The results of Real-time PCR showed that R2I4R2 and K2I4K2 increased the amplification efficiency of Taq enzyme by about 1.75 and 1.86 times. Conclusion: the novel self-assembled peptide R2I4R2 and K2I4K2 are stable in secondary structure and can be used in three-dimensional cell culture. Both peptides can stimulate the regeneration of blood vessels and promote the migration of fibroblasts to repair skin trauma. This study will stimulate the development of short peptide design concept and the application of short peptide in tissue repair and regeneration, protein stabilization and so on.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R641
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