新型自組裝短肽對皮膚創(chuàng)傷快速修復(fù)過程研究
發(fā)布時間：2019-03-01 21:28
【摘要】：目的:采用新型自組裝短肽R2I4R2和K2I4K2應(yīng)用于細胞三維培養(yǎng)和皮膚創(chuàng)傷快速修復(fù),并初步探索其作用機制。方法:剛果紅染色和原子力顯微鏡(AFM)檢測短肽R2I4R2和K2I4K2的宏觀結(jié)構(gòu);圓二色光譜(CD)檢測理化條件對短肽R2I4R2和K2I4K2二級結(jié)構(gòu)的影響;紅細胞裂解試驗檢測兩條短肽對細胞膜的裂解作用;CCK-8和AO/EB染色檢測短肽R2I4R2和K2I4K2對人皮膚成纖維細胞HFF-1在三維環(huán)境中增殖情況;流式細胞周期分析HFF-1細胞在短肽形成的三維體系中的周期變化;建立SD大鼠皮膚創(chuàng)傷修復(fù)模型,考察兩條短肽對皮膚創(chuàng)傷修復(fù)的影響;HE染色和免疫組化檢測短肽R2I4R2和K2I4K2對皮膚創(chuàng)傷修復(fù)過程的病理變化;Real-time PCR檢測皮膚修復(fù)相關(guān)蛋白基因和通路相關(guān)基因表達量的變化;以Taq DNA聚合酶作蛋白穩(wěn)定假設(shè)模型,通過紫外可見光譜和real-time PCR考察短肽R2I4R2和K2I4K2對蛋白分子的保護作用。結(jié)果:CD檢測顯示兩條短肽都具有介于α-螺旋和無規(guī)卷曲之間的二級結(jié)構(gòu),且在不同的離子和溫度條件下都能保持較為穩(wěn)定的結(jié)構(gòu)。剛果紅染色和(AFM)結(jié)果表明兩條短肽都能自組裝形成納米纖維支架,進而形成水凝膠。而紅細胞裂解試驗和CCK-8檢測結(jié)果顯示兩條短肽均無細胞毒性,能夠很好的應(yīng)用于細胞三維培養(yǎng)和創(chuàng)傷修復(fù)。動物實驗表明短肽R2I4R2使創(chuàng)傷修復(fù)速度提高約28%,而K2I4K2提高約34%。而HE染色結(jié)果顯示皮膚傷口加入短肽后3-5 d炎癥細胞減少,7 d有血管形成,15 d后肉芽組織纖維化,進入組織重塑期。免疫組化結(jié)果進一步明確在加入短肽R2I4R2和K2I4K2后,5 d時血管內(nèi)皮細胞因子CD34減少,7 d時VEGF含量增多。Real-time PCR結(jié)果顯示HFF-1細胞在短肽形成的三維體系中,PEGRF基因表達量降低,VEGF、Integrin-α3、Integrin-α5和β-tublin基因表達量都有明顯升高。UV檢測結(jié)果顯示,短肽R2I4R2和K2I4K2使對假設(shè)模型分子Taq酶在高溫下的半衰期提高了1.44和1.68倍。Real-time PCR結(jié)果表明短肽R2I4R2和K2I4K2使Taq酶的擴增效率提高了約1.75和1.86倍。結(jié)論:本研究采用的新型自組裝短肽R2I4R2和K2I4K2二級結(jié)構(gòu)穩(wěn)定,都能應(yīng)用于細胞的三維培養(yǎng)。且兩條短肽都能刺激創(chuàng)傷處血管再生、促進成纖維細胞遷移而使皮膚創(chuàng)傷快速修復(fù)的作用。本研究將激發(fā)更多的短肽設(shè)計理念的開發(fā)和短肽在組織修復(fù)再生、穩(wěn)定蛋白質(zhì)等領(lǐng)域中的應(yīng)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 偽-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- 偽 3, Integrin- 偽 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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R641
本文編號：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 偽-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- 偽 3, Integrin- 偽 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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R641
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