聚乙二醇修饰的成纤维细胞生长因子-21的降血糖作用

The Hypoglycemic Effect of The PEGylated FGF-21

成纤维细胞生长因子-21(FGF-21)是FGF家族的一员.现有大量研究表明,FGF-21是除胰岛素以外的一种新的血糖调节因子,有望成为治疗2型糖尿病的新型药物.然而,FGF-21在动物体内稳定性较差,半衰期较短,严重影响了其在临床上的应用.为解决这些问题,本实验采用分子质量为20 ku的单甲氧基聚乙二醇-丙醛(mPEG-ALD)对鼠源FGF-21(mFGF-21)进行N端定点修饰,以改善mFGF-21的性质(如提高体内半衰期、降低免疫原性等).本文研究了反应pH、反应时间、蛋白质浓度及反应物之间的质量比对mFGF-21与聚乙二醇(PEG)合成反应的影响.采用Capto Q阴离子交换层析或Superdex 75凝胶过滤层析分离纯化聚乙二醇化mFGF-21(PEG-mFGF-21),并最终确定了mFGF-21聚乙二醇修饰的反应条件和分离PEG-mFGF-21的纯化工艺.随后分别进行了PEG-mFGF-21的理化性质(大小、纯度和体外稳定性)、免疫原性、体内半衰期、体外葡萄糖吸收活性及体内降糖活性的研究.体外稳定性实验结果显示,mFGF-21经PEG修饰后温度稳定性和抗蛋白酶水解稳定性都显著提高.间接ELISA方法检测血清中mFGF-21抗体水平及目标蛋白含量的结果表明,PEG修饰mFGF-21可明显降低其免疫原性,延长体内半衰期.HepG2细胞的葡萄糖吸收实验结果发现,PEG-mFGF-21的细胞活性并没有下降,反而随着刺激细胞时间的延长,经PEG-mFGF-21刺激的细胞葡萄糖吸收显著高于mFGF-21刺激的细胞葡萄糖吸收.2型糖尿病db/db小鼠短期血糖调控实验结果表明,mFGF-21降糖速度快于PEG-mFGF-21,但其持续时间较PEG-mFGF-21短;长期血糖调控实验结果显示,PEG-mFGF-21长期降糖效果优于mFGF-21,作用持续时间长,并且PEG-mFGF-21在停药后控制血糖的能力也高于mFGF-21.综上所述可知,mFGF-21的PEG修饰在不影响其体外生物活性的前提下,能够提高mFGF-21的物理稳定性和抵抗蛋白酶水解的能力、降低免疫原性、增加体内稳定性、延长mFGF-21在动物体内降血糖作用的效果和时间.本实验为FGF-21化学修饰提供了重要的技术平台,对以后FGF-21的临床应用具有非常重要的意义.

Fibroblast growth factor 21（FGF-21） is a member of FGF family.Recently,it is discovered as a non-insulin-dependent cytokine to regulate blood glucose and a potential drug candidate for treatment of type 2 diabetes mellitus.However,the stability of FGF-21 is poor and its half-life in vivo is short,which severely affect its application in clinical practice.In order to solve this problem,the N-terminus of mFGF-21 was PEGylated in a site-specific manner by methoxy poly-ethylene glycol（mPEG） propionaldehydes with an average molecular mass of 20 ku for improving its biological properties including increasing half-life in vivo,and decreasing immunogenicity.The effects of pH,reaction time,protein concentrations and mass ratio between the reactants on the PEGylation of mFGF-21 were analyzed.PEG-mFGF-21 was isolated by Capto Q anion exchange chromatography or Superdex 75 gel filtration chromatography.As a result,the optimal reaction method for mFGF-21 PEGylation and purification processes of PEG-mFGF-21 were established.Then we study the physical and chemical properties,immunogenicity,in vivo half-life,in vitro biological activity and in vivo hypoglycemic effects of PEG-mFGF-21.We found that the temperature stability and anti-protease ability of mFGF-21 were significantly improved after PEGylation.The indirect ELISA results for detection of serum antibody levels against mFGF-21 and the target protein concentration showed that PEGlyation of mFGF-21 significantly reduced its immunogenicity and increased its half-life in vivo.The glucose uptake assay results in HepG2 cells demonstrated that the in vitro activity of PEG-mFGF-21 did not decline.However,with the increment of stimulating time,the glucose uptake in cells treated with PEG-mFGF-21 significantly increased than mFGF-21.The experimental results of short-term blood glucose regulation in type 2 diabetic animals showed that the hypoglycemic speed of mFGF-21 was faster than PEG-mFGF-21,but it lasted shorter.The long-term blood glucose regulation experimental results showed that PEG-mFGF-21 had better hypoglycemic effect than mFGF-21.Surprisingly,blood glucose of PEG-mFGF-21-treated mice remained at low level for several days after the drug was withdrawn.In conclusion,under the premise of remaining its biological activity in vitro,PEGylation of mFGF-21 can improve its physical stability and anti-protease ability,decrease its immunogenicity and increase its in vivo half-life.It can also prolong the hypoglycemic effect of mFGF-21 in diabetic animals.This study provides an important technology platform for drug development of FGF-21.