多肽促人表皮生长因子透皮输运机理及其应用研究

发布时间：2018-04-03 06:18

本文选题：透皮多肽　切入点：表皮生长因子　出处：《中国科学技术大学》2015年博士论文

【摘要】：皮肤为全身给药提供了方便的界面,然而由于角质层形成的皮肤屏障,绝大部分蛋白质类亲水性药物分子通常很难透过皮肤。生物来源的多肽,比如TD1和SPACE,已经被证明能够通过局部混合给药方式促进多种生物大分子经皮渗透,但目前多肽增强大分子药物透皮机理尚未完全清楚。本研究中,我们进一步拓展了多肽的应用并研究了多肽促透机制。本文工作对研发新型透皮肽以提高蛋白类药物经皮给药效率具有重要的理论和应用意义。首先,我们基于TD1设计了一个新的透皮药物,即人表皮生长因子(hEGF)与透皮肽TD1的融合蛋白：TD1-hEGF。研究表明TD1-hEGF不仅具有和天然hEGF相同的生物学活性,还拥有相当强的透皮能力,并且比TD1和hEGF混合透皮给药效率更高。将TD1-hEGF联合抗菌药物用于治疗念珠菌性阴道炎,不仅可以有效提高抗菌药的治疗效果,同时还能有效修复阴道外层粘膜。其次,我们发现了TD1-hEGF的促透过程具有能量依赖性。研究结果显示,TD1-hEGF的透皮活性会被能量抑制剂和温度所降低,而三磷酸腺苷(ATP)能更有效促进TD1-hEGF的透皮,并表现浓度依赖性。这种能量依赖性在人类皮肤上也得到了验证。此外,我们还建立了一个简单的数学模型模拟了TD1参与的hEGF透皮运输过程。第三,我们发现了钠钾ATP酶β亚基(ATP1B1)在TD1介导的药物透皮过程中具有重要作用。通过酵母双杂交系统和免疫共沉淀实验,我们筛选并确认了在酵母和哺乳动物细胞中ATP1B1的C端和TD1具有特异的亲和能力。研究显示TD1和ATP1B1的相互作用不仅影响了ATP1B1的表达,同时还影响了其在细胞中的定位。这种相互作用会被外源竞争物或ATP1B1抑制剂所抵抗,从而减弱大分子药物的透皮。最后,我们研究了将SPACE和hEGF的融合蛋白用于携带siRNA经皮靶向诱导皮肤黑色素瘤细胞凋亡。通过体外实验已证明SPACE-hEGF具有透皮和穿细胞功能,还能够通过hEGF高效靶向到EGFR过表达的黑色素瘤细胞上,SPACE-hEGF装载了GAPDH和c-Myc siRNA后有效降低了相关基因的转录水平,并诱导了黑色素细胞凋亡的产生。
[Abstract]:Skin provides a convenient interface for systemic drug delivery. However, because of the skin barrier formed by the cuticle, it is usually difficult for most protein hydrophilic drug molecules to penetrate the skin.Biologically derived peptides, such as TD1 and spa, have been shown to be able to promote the transdermal permeation of many biological macromolecules through local administration. However, the mechanism of polypeptides enhancing the transdermal penetration of macromolecular drugs has not been fully understood.In this study, we further expanded the application of polypeptides and studied the mechanism of polypeptide penetration.This work has important theoretical and practical significance for the development of new transdermal peptides to improve the transdermal delivery efficiency of protein drugs.Firstly, we designed a new transdermal drug based on TD1, the fusion protein of human epidermal growth factor hEGFand transdermal peptide TD1: TD1-hEGF.The results showed that TD1-hEGF not only had the same biological activity as natural hEGF, but also had strong transdermal ability, and had higher transdermal delivery efficiency than TD1 and hEGF.The use of TD1-hEGF combined with antibiotics in the treatment of Candida vaginitis can not only effectively improve the therapeutic effect of antibiotics, but also effectively repair the outer mucosa of vagina.Secondly, we found that the permeation process of TD1-hEGF is energy dependent.The results showed that the transdermal activity of TD1-hEGF was decreased by energy inhibitor and temperature, while adenosine triphosphate (ATP) was more effective in promoting the transdermal permeability of TD1-hEGF in a concentration-dependent manner.This energy dependence has also been demonstrated in human skin.In addition, a simple mathematical model is established to simulate the hEGF transdermal transport process in which TD1 is involved.Thirdly, we found that ATP-1B1 of sodium and potassium ATP enzyme 尾 subunit plays an important role in TD1 mediated drug transdermal process.By yeast two-hybrid system and co-immunoprecipitation, we have screened and confirmed the specific affinity between C-terminal and TD1 of ATP1B1 in yeast and mammalian cells.The results showed that the interaction of TD1 and ATP1B1 not only affected the expression of ATP1B1, but also affected its localization in cells.This interaction is resisted by exogenous competition or ATP1B1 inhibitors, which weakens the transdermal penetration of macromolecular drugs.Finally, we studied the application of SPACE and hEGF fusion protein to induce apoptosis of skin melanoma cells by transcutaneous targeting with siRNA.It has been proved by in vitro experiments that SPACE-hEGF has transdermal and transdermal functions, and can efficiently target to EGFR over-expressed melanoma cells by hEGF. SPACE-hEGF can effectively reduce the transcription level of related genes by loading GAPDH and c-Myc siRNA into melanoma cells.The apoptosis of melanocytes was induced.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R943

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