黏附性胰岛素样生长因子-1的Sortase A酶法制备以及其对医用金属钛材料表面修饰的应用

Medical titanium material has good mechanical properties and biocompatibility, but its surface bioinsert has limited its application in medicine. It is suggested that bio-chem orthogonal protein engeering can be applied to endow the surface bioactivity of metal materials, thus solving the defects of metal material surface inertia. Inspired by bionic characteristics of marine mussel foot protein, using 3,4-L-dihydroxyphenylalanine (DOPA) as media, prepare for human insulin-like growth factor-1 (IGF-1) through a bioorthogonal approach of bioengineering and chemical enzyme engineering, then use adhesive IGF-1 to modify medical titanium material surface. Modified titanium material surface has the biological activity of IGF-1. In previous study, data showed it can promote cells proliferation on material surface after adhesive IGF-1 immobilization though Tyrosinase synthetic method, however, the effect in vivo is still unknow.The disadvantages of Tyrosinase synthetic method is modification of 3 Tyr in IGF-1 and the products is uncontrol. This project will obtain adhesive IGF-1 by Sortase A synthetic method and implant immobilzed titanium material into bone defect animal model and observe the influence of cell proliferation and tissue repairment. It will improve the method to solve the bioinnert defect of medical titanium surface and obtain bioactivity titanium material, and it may also provide new approach and new idea for developing a biological functional titanium material to meet the needs of clinical application.

医用钛金属材料因表面惰性问题限制了医学上的应用，提出用生物化学正交蛋白质工程理念解决金属材料表面惰性的缺陷。受海洋贻贝黏附性丝足蛋白的启发，以DOPA作为媒介，通过生物化学正交蛋白质工程方法，制备出具有黏附性的IGF-1，并利用黏附性IGF-1对钛进行表面修饰，赋予修饰后钛表面具有IGF-1的生物学活性。前期研究结果提示，以Tyrosinase酶法合成的黏附性IGF-1修饰钛后，可促进体外细胞的增殖，但体内作用效果尚不明确。该合成法的不足是：改变IGF-1天然序列中的3个Tyr和反应产物不可控。根据前期结果及存在问题，本项目提出利用Sortase A酶法合成黏附性IGF-1，拟将黏附性IGF-1修饰后的钛移植到动物骨缺损模型中，探究其在体内对细胞增殖及组织修复的影响。该研究将为解决医用钛材料表面生物惰性的缺陷提供新方法，制备出具有生物活性的钛，满足钛在临床应用上对生物学活性的需求。

为解决医用钛材料表面生物惰性问题，本项目采用生物工程方法通过贻贝丝足粘附蛋白将IGF-1修饰于钛材料表面，赋予钛表面IGF-1的生物学活性同时将IGF-1固定在介质表面，使其缓慢且长期产生信号传导作用，并进行体内骨缺损修复的疗效评价。前期研究结果提示，以Tyrosinase酶法合成的黏附性IGF-1修饰钛后，可促进体外细胞的增殖，但体内作用效果尚不明确。该合成法的不足是：改变IGF-1天然序列中的3个Tyr和反应产物不可控。根据前期结果及存在问题，本项目提出利用Sortase A酶法合成黏附性IGF-1，将黏附性IGF-1修饰后的支架移植到动物骨缺损模型中，探究其在体内对细胞增殖及组织修复的影响。MALDI-TOF/MS结果提示Sortase A酶法制备黏附性IGF-1的可行性；通过3种不同细胞对固定化IGF-1的活性进行检测，结果提示固定化IGF-1具有更高的细胞增殖促进作用；利用NBT实验、Anti-IGF-1实验和共聚焦显微镜检测黏附性IGF-1的粘附效果，结果均说明带有贻贝仿生黏附性接头多肽XKXKX的IGF-1具有更强的材料表面黏附性。体内实验方面，我们做了大鼠颅骨缺损模型，并对黏附性IGF-1和非黏附性IGF-1修饰的支架进行8周修复实验，结果提示，黏附性IGF-1修饰支架具有更好的修复效果。值得一提的是，项目进行中还融入了增材制造技术来制备个性化骨科植入用支架。本项目将为活性钛材料的制备提供新思路与新方法，具有良好的临床应用前景。