重组人骨形态发生蛋白-2的制备及成骨活性表征  

Preparation of recombinant human bone morphogenetic protein-2 and investigation of osteogenic activity

作　　者：李贵龙[1] 王靖[1] 刘昌胜[1] 

机构地区：[1]华东理工大学教育部医用生物材料工程研究中心,上海200237

出　　处：《化工学报》2015年第8期3183-3188,共6页CIESC Journal

基　　金：国家重点基础研究发展计划项目(2012CB933600)~~

摘　　要：骨形态发生蛋白-2(BMP-2)是重要的骨诱导生长因子,是提高骨修复材料活性和临床骨修复效果的有效手段和关键物质。由于BMP-2在体内含量低,依靠从动物体内提取难以满足临床需求。本文研究满足临床需求的BMP-2的制备方法,并评价其生物活性。采用密码子优化方法,并通过进一步更换其中部分核苷酸编码,得到优化的h BMP-2基因的DNA序列,制备大肠杆菌rh BMP-2菌株,通过发酵及工艺优化,获得BMP包涵体,经分离纯化与复性,制备出高纯度的rh BMP-2。测定C2C12细胞的碱性磷酸酶(ALP)活性来表征单倍体和二倍体BMP-2的成骨活性,发现二倍体rh BMP-2的成骨活性明显高于单倍体rh BMP-2,并且随着BMP-2浓度增加,碱性磷酸酶活性上升。体内动物异位成骨实验发现rh BMP-2肌带植入小鼠体内3周后,取出的异位骨颗粒鲜艳饱满,骨结构完整;HE切片和Masson三色切片都显示出良好的异位成骨效果。此方法制备的rh BMP-2具有良好的诱导成骨分化能力,可用于骨组织修复,满足临床需要。Bone morphogenetic protein-2 （BMP-2）, one of the important osteoinductive growth factors, is an effective and crucial cytokine for clinic bone regeneration as well as bioactivity improvement of bone substitute. However, extraction of BMP-2 from animals can hardly meet the clinic requirements due to its very low content. This work studied the preparation of BMP-2 to meet the clinical needs, and evaluated its biological activity. Firstly, DNA sequence of optimized hBMP-2 gene was obtained to prepare rhBMP-2 strains ofE.coli using codon optimization method with further replacement of the partial nucleotide coding. Then, through fermentation and process optimization the BMP inclusion body was acquired. After separation, purification and renaturation, rhBMP-2 of high purity was prepared.In vitro cell experiments, the activity of alkaline phosphatase （ALP） in C2C12 cells was measured to characterize the osteogenic activity of haploid and diploid BMP-2. It was observed that the osteogenic activity of diploid rhBMP-2 was significantly higher than that of haploid rhBMP-2, and the activity of alkaline phosphatase increased with the increase of the BMP-2 concentration.In vivo ectopic bone experiment, after rhBMP-2 was implanted into the muscle of mice for 3 weeks, the ectopic bone granules were＆nbsp;bright and fresh, and the bone structure was complete. HE and Masson’s trichrome stainings showed good ectopic bone formation. It can be concluded that the rhBMP-2 prepared using this method can well induce osteogenic differentiation, and can be used for bone tissue repair to meet the clinical needs.

关 键 词：骨修复 生物医学工程 蛋白质 蛋白质复性 骨形态发生蛋白-2 原核表达 

分 类 号：R318.08[医药卫生—生物医学工程]