2型糖尿病多肽药物利拉鲁肽纳米制剂的制备

发布时间：2018-03-22 04:22

本文选题：2型糖尿病　切入点：胰高血糖素样肽1　出处：《湖北工业大学》2017年硕士论文　论文类型：学位论文

【摘要】：2型糖尿病是最为常见的糖尿病类型,占糖尿病患者人数90%以上。胰高血糖素样肽1(GLP-1)是一种小肠L型细胞所释放的的天然肠促胰岛素激素,能有效降低血糖,促进胰腺β细胞增殖,防止β细胞凋亡和延迟胃排空,在2型糖尿病的治疗中具有良好的前景。然而,天然GLP-1在体内易被二肽基肽酶4(DPP-4)降解,使半衰期仅为1至2分钟,限制其临床上的直接应用,因此开发更稳定的GLP-1类似物或缓释制剂成为了近年来的研究热点。利拉鲁肽是销量最好的酰化的人GLP-1类似物之一,与内源性人GLP-1(7-37)具有97%的同源性,并具有良好的降糖效应。虽然利拉鲁肽的半衰期可达13小时,但仍需要一天注射一次,为了提高患者的顺应性和提高利拉鲁肽的疗效,因此有必要研究长效化多肽药物。我们旨在制备利拉鲁肽纳米制剂,先通过基因工程制备利拉鲁肽的前体,再通过化学修饰制备利拉鲁肽,最后通过纳米粒包埋利拉鲁肽。利拉鲁肽的前体,即GLP-1~(R34),多使用多肽固相合成方法以制备利拉鲁肽前体。而在我们的研究中应用了一种新方法,其中包括在大肠杆菌中将MFH-GLP-1~(R34)融合蛋白通过乳糖诱导并以包涵体形式表达,GLP-1~(R34)的纯化以及在GLP-1~(R34) 26号位通过谷氨酸介导接上棕榈酸侧链。该载体蛋白有助于利拉鲁肽前体的过表达,与IPTG诱导的表达相比,乳糖诱导的表达对细胞显示无毒性作用,且表达量相当。通过优化乳糖浓度、诱导时间、初始诱导细胞光密度值和培养基条件进行发酵罐培养融合蛋白,并通过分批补料最终获得了10 g/L的菌体量。通过离子交换色谱法成功地纯化了利拉鲁肽前体,获得90%以上的纯度,并通过HPLC进行分离。我们的研究结果表明,通过离子交换制备利拉鲁肽前体的工艺是经济又有效的。通过乳化溶剂蒸发法制备了聚乙醇酸羟基乙酸(PLGA)纳米粒,并优化了制备PLGA纳米粒的工艺,包括溶剂、乳化剂浓度、稀释比例,最终得到了均匀、粒径小、稳定性高的纳米粒,并将其用于后续生物学活性实验。综上,本研究采用大肠杆菌作为利拉鲁肽前体表达的宿主菌,相比传统方法中在酿酒酵母中表达利拉鲁肽前体要更加经济,采用离子交换方法纯化利拉鲁肽前体要更加简便高效,并探索了PLGA纳米粒的制备工艺,为长效GLP-1类似物药物的开发奠定了基础。
[Abstract]:Type 2 diabetes is the most common type of diabetes, accounting for more than 90% of the patients with diabetes. Glucagon like peptide 1 (GLP-1) is a natural intestinal insulin stimulating hormone released by L-type cells of the small intestine, which can effectively reduce blood glucose. Promoting pancreatic 尾 cell proliferation, preventing 尾 cell apoptosis and delayed gastric emptying have good prospects in the treatment of type 2 diabetes. However, natural GLP-1 is easily degraded by dipeptidyl peptidase 4 (DPP-4) in vivo, and the half-life is only 1 to 2 minutes. Therefore, the development of more stable GLP-1 analogues or slow-release preparations has become a hot topic in recent years. Lilaru peptide is one of the best-selling acylated human GLP-1 analogues, and has 97% homology with endogenous human GLP-1 7-37). And has a good hypoglycemic effect. Although the half-life of liraropeptide can be up to 13 hours, it still needs to be injected once a day, in order to improve the compliance of patients and improve the curative effect of liralutin. Therefore, it is necessary to study long-acting polypeptide drugs. Finally, a new method was applied in our research to prepare the precursor of Lilaru peptide, which is the precursor of Lilaru peptide, GLP-1, R34, which was encapsulated by nanocrystalline particles, and the solid phase synthesis method of polypeptide was used to prepare the precursor of Lilaru peptide. These include the purification of MFH-GLP-1 / R34) fusion protein induced by lactose and expressed as inclusion body in Escherichia coli, and the incorporation of palmitic acid side chain via glutamic acid mediated at GLP-1 / R34). The carrier protein contributes to the overexpression of Lilaru peptide precursor, Compared with the expression induced by IPTG, the expression induced by lactose showed no toxicity to cells, and the expression level was equal. The fusion protein was cultured in fermentor by optimizing the concentration of lactose, inducing time, initial optical density of induced cells and medium condition. Finally, 10 g / L biomass was obtained by batch feeding. The precursor was purified successfully by ion exchange chromatography, the purity was over 90%, and the purity was separated by HPLC. Our results show that, It is economical and effective to prepare the precursor of Lilaru peptide by ion exchange. Polyglycolic acid (PLGA) nanoparticles were prepared by emulsifying solvent evaporation method, and the process of preparing PLGA nanoparticles, including solvent and emulsifier concentration, was optimized. Finally, homogenous, small and stable nanoparticles were obtained by dilution ratio, and were used in the subsequent biological activity experiments. In summary, Escherichia coli was used as the host strain for the expression of Lilaru peptide precursor. Compared with the traditional methods, it is more economical to express the precursor of Lilaru peptide in Saccharomyces cerevisiae, and it is more convenient and efficient to purify the precursor of Lilaru peptide by ion exchange method, and the preparation process of PLGA nanoparticles is explored. It lays a foundation for the development of long-acting GLP-1 analogues.
【学位授予单位】：湖北工业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TQ464.7

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