[1]涂兵,叶吉星,甘翼搏,等.碱性成纤维细胞生长因子和骨形态发生蛋白在Origami2体系中的可溶表达及二者促成骨的协同效应[J].第三军医大学学报,2018,40(10):882-888.
 TU Bing,YE Jixing,GAN Yibo,et al.Soluble expression of basic fibroblast growth factor and bone morphogenetic protein in Origami2 system and their synergistic effect to promote osteogenesis[J].J Third Mil Med Univ,2018,40(10):882-888.
点击复制

碱性成纤维细胞生长因子和骨形态发生蛋白在Origami2体系中的可溶表达及二者促成骨的协同效应(/HTML )
分享到:

《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
40卷
期数:
2018年第10期
页码:
882-888
栏目:
基础医学
出版日期:
2018-05-30

文章信息/Info

Title:
Soluble expression of basic fibroblast growth factor and bone morphogenetic protein in Origami2 system and their synergistic effect to promote osteogenesis
作者:
涂兵叶吉星甘翼搏张立泰欧阳斌罗向东周强
陆军军医大学(第三军医大学)第一附属医院:全军矫形外科中心,组织工程国家地方联合工程实验室,重庆市再生重建医学工程技术研究中心,重庆市组织工程实验室,全军烧伤研究所,创伤、烧伤与复合伤国家重点试验室
Author(s):
TU Bing YE Jixing GAN Yibo ZHANG Litai OUYANG Bin LUO Xiangdong ZHOU Qiang

Department of Orthopedics, National & Regional United Engineering Laboratory of Tissue Engineering, 2Stace Key Laboratory of Trauma, Burns and Combined Injury, Institution of Burn Research, First Affiliated Hospital, Army Medical University (Third Military Medical University), Chongqing, 400038, China

关键词:
碱性成纤维细胞生长因子骨形态发生蛋白2基因重组协同效应成骨
Keywords:
basic fibroblast growth factor bone morphogenetic protein genetic recombination synergistic effect osteogenesis
分类号:
R322.71;R39433;R394.3
文献标志码:
A
摘要:

目的    建立优化高效的碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)、骨形态发生蛋白2(bone morphogenetic protein,BMP-2)蛋白重组方法,阐明二者在促成骨中的协同效应。方法    优化bFGF和BMP-2成熟肽的基因序列密码子,获得适用于大肠杆菌Origami2体系表达的bFGF及BMP-2成熟肽cDNA片段;利用PCR法将bFGF成熟肽cDNA片段插入原核表达质粒pColdⅡ中,构建重组原核表达质粒pColdⅡ-bFGF;利用PCR法将BMP-2成熟肽cDNA片段插入原核表达质粒pET-30a(+)中,构建重组原核表达质粒pET-30a(+)BMP-2,转入大肠杆菌Origami2中表达纯化;运用双酶切、质粒测序及Western blot技术证明Origami2表达体系的构建效果;进行细胞增殖实验、碱性磷酸酶(alkaline phosphates,ALP)染色观察和体内成骨效率检测等,探索bFGF、BMP-2在促成骨中的协同效应。结果    双酶切和质粒测序鉴定证明成功构建重组大肠杆菌pColdⅡbFGF/Origami2和pET30a(+)BMP-2/Origami2表达体系,Western blot检测发现蛋白正确表达。经过NiNTA亲和层析纯化后bFGF及BMP-2纯度高达90%。通过对二者比例的优化筛选,发现40 ng/mL bFGF+40 ng/mL rhBMP-2时,大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)增殖效率、ALP活性及体内成骨效率最高,是二者促成骨的最优效组合。结论    成功构建高效表达的pColdⅡ-bFGF/Origami2和pET30a(+)BMP-2/Origami2表达体系,实现了bFGF及BMP-2的高效表达。bFGF和BMP-2的优化联合应用具有促成骨协同效应。

Abstract:

Objective    To establish recombinant expression systems for basic fibroblast growth factor (bFGF) and bone morphogenetic protein (BMP-2) and investigate the synergetic effect of bFGF and BMP-2 in promoting osteogenesis. Methods    The codons encoding mature peptide sequences of bFGF and BMP-2 gene were optimized and their cDNA fragments capable of expressions in E.coli Origami2 system were obtained. Using PCR method, the mature peptide cDNA fragments of bFGF and BMP-2 were separately inserted in the prokaryotic expression plasmids pColdⅡ and pET-30a (+) to construct the recombinant expression plasmids pColdⅡ-bFGF and pET-30a (+)BMP-2, respectively. The plasmids were transferred into E.coli Origami2 for expression and the products were purified. Double enzyme digestion, sequencing analysis and Western blotting were used to verify the established Origami2 expression systems. The synergetic effect of the recombinant bFGF and BMP-2 proteins in promoting osteogenesis was tested by observing their effect on proliferation of rat one marrow mesenchymal stem cells (BMSCs), ALP activity in mouse C2C12 myoblasts and osteogenesis of BMSCs in rat quadriceps femoris in vivo. Results    Double enzyme digestion and sequencing analysis verified successful construction of recombinant E.coli pColdⅡ-bFGF/Origami2 and pET30a (+)BMP-2/Origami2 expression systems. Western blotting demonstrated expressions of the two target proteins in the expression systems, and after purification with Ni-NTA affinity chromatography, the expressed bFGF and BMP-2 proteins reached a purity as high as 90%. At the optimized ratio, bFGF (40 ng/mL) combined with rhBMP2 (40 ng/mL) obviously promoted the proliferation of rat BMSCs, enhanced ALP activity in C2C12 myoblasts, and efficiently promoted ectopic osteogenesis of BMSCs in rat quadriceps femoris. Conclusion    We successfully constructed pColdⅡ-bFGF/Origami2 and pET30a (+)BMP-2/Origami2 expression systems for efficient expression of bFGF and BMP-2. At the optimized ratio, bFGF and BMP-2 produce a synergistic effect to promote osteogenesis of BMSCs.

参考文献/References:

[1]朱璨, 节云峰, 张倩, 等. 多孔钽颗粒负载碱性成纤维细胞生长因子促进犬颌骨缺损修复的初步研究[J]. 第三军医大学学报, 2016, 38(11): 1240-1244. DOI: 10.16016 /j.1000-5404.201511032.
ZHU C, JIE Y F, ZHANG Q, et al. Primary study on repair of canine jaw defects with porous tantalum particles loaded with basic fibroblast growth factor[J]. J Third Mil Med Univ, 2016, 38(11): 1240-1244. DOI: 10.16016 /j.1000-5404.201511032.
[2]CHEN B, HE J, YANG H, et al. Repair of spinal cord injury by implantation of bFGF-incorporated HEMA-MOETACL hydrogel in rats[J]. Sci Rep, 2015, 5: 9017. DOI: 10.1038/srep09017.
[3]WANG F, XU R, XU Q, et al. Effect of laser therapy on plasma expression of VEGF and bFGF in infants with cutaneous hemangioma [J]. Oncol Lett, 2017, 13(3): 1861-1865. DOI: 10.3892/ol.2017.5640.
[4]KATAGIRI T, YAMAGUCHI A, KOMAKI M, et al. Bone morphogenetic protein2 converts the differentiation pathway of C2C12 myoblasts into the osteoblast lineage[J]. J Cell Biol, 1994, 127(6 Pt 1): 1755-1766.
[5]CHURCH R H, KRISHNAKUMAR A, URBANEK A, et al. Gremlin 1 preferentially binds to bone morphogenetic protein-2 (BMP-2) and BMP-4 over BMP-7[J]. Biochem J, 2015, 466(1): 55-68.DOI: 10.1042/BJ20140771.
[6]DRAGOO J L, CHOI J Y, LIEBERMAN J R, et al. Bone induction by BMP-2 transduced stem cells derived from human fat[J]. J Orthop Res, 2003, 21(4): 622-629. DOI: 10.1016/S0736-0266(02)00238-3.
[7]LUCA L, ROUGEMONT A L, WALPOTH B H, et al. Injectable rhBMP-2-loaded chitosan hydrogel composite: osteoinduction at ectopic site and in segmental long bone defect[J]. J Biomed Mater Res A, 2011, 96(1): 66-74. DOI: 10.1002/jbm.a.32957.
[8]DU M, ZHUHU T, DUAN X, et al. Acellular dermal matrix loading with bFGF achieves similar acceleration of bone regeneration to BMP-2 via differential effects on recruitment, proliferation and sustained osteo differentiation of mesenchymal stem cells[J]. Mater Sci Eng C Mater Biol Appl, 2017, 70(Pt 1): 62-70. DOI: 10.1016/j.msec.2016.08.049.
[9]QI W, YAN J, SUN H, et al. Multifunctional Nanocomposite Films for Synergistic Delivery of bFGF and BMP-2[J]. ACS Omega, 2017, 2(3): 899-909. DOI: 10.1021/acsomega.6b00420.
[10]CONNELLY J T, GARCIA A J, LEVENSTON M E.Interactions between integrin ligand density and cytoskeletal integrity regulate BMSC chondrogenesis[J]. J Cell Physiol, 2008, 217(1): 145-154. DOI: 10.1002/jcp.21484.
[11]HANADA K, SOLCHAGA L A, CAPLAN A I, et al. BMP-2 induction and TGF-beta 1 modulation of rat periosteal cell chondrogenesis[J]. J Cell Biochem, 2001, 81(2): 284-294.
[12]DUAN Z X, ZHENG Q X, GUO X D.Experimental study of BMP-2 derived peptide on osteogenic induction in BMSC in vitro[J]. Orthopedic J Chin, 2007.
[13]LIU Y, YI X C, GUO G, et al. Basic fibroblast growth factor increases the transplantationmediated therapeutic effect of bone mesenchymal stem cells following traumatic brain injury[J]. Mol Med Rep, 2014, 9(1): 333-339. DOI: 10.3892/mmr.2013.1803.
[14]WANG L, HUANG Y, PAN K, et al. Osteogenic responses to different concentrations/ratios of BMP-2 and bFGF in bone formation[J]. Ann Biomed Eng, 2010, 38(1): 77-87. DOI: 10.1007/s10439-009-9841-8.
[15]FUJIOKAKOBAYASHI M, OTA M S, SHIMODA A, et al. Cholesteryl group and acryloyl groupbearing pullulan nanogel to deliver BMP2 and FGF18 for bone tissue engineering[J]. Biomaterials, 2012, 33(30): 7613-7620. DOI: 10.1016/j.biomaterials.2012.06.075.
[16]LIN C Y, CHANG Y H, LI K C, et al. The use of ASCs engineered to express BMP2 or TGF-β3 within scaffold constructs to promote calvarial bone repair[J]. Biomaterials, 2013, 34(37): 9401-9412. DOI: 10.1016/j.biomaterials.2013.08.051.
[17]VOGT S, WEXEL G, TISCHER T, et al. The influence of the stable expression of BMP2 in fibrin clots on the remodelling and repair of osteochondral defects[J]. Biomaterials, 2009, 30(12): 2385-2392. DOI: 10.1016/j.biomaterials.2009.01.016.
[18]李素铠, 欧阳斌, 徐晓秋, 等.PLGA纳米药物递送系统的建立及其对bFGF生物学活性的维持[J]. 第三军医大学学报, 2016, 38(11): 1319-1324. DOI: 10.16016 /j.1000-5404.201601019.
LI S K, OUYANG B, XU X Q, et al. Establishment of poly(DL-lactide-co-glycolide) nanoparticle drug delivery system and its maintenance for bioactivity of basic fibroblast growth factor[J]. J Third Mil Med Univ, 2016, 38(11): 1319-1324. DOI: 10.16016 /j.1000-5404.201601019.
[19]BAI Y, LI P, YIN G, et al. BMP-2, VEGF and bFGF synergistically promote the osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells[J]. Biotechnol Lett, 2013, 35(3): 301-308. DOI: 10.1007/s10529-012-1084-3.

相似文献/References:

[1]周栩,权正学,罗小辑,等.Id1基因在BMP-2维持椎间盘细胞软骨特性中的作用[J].第三军医大学学报,2015,37(20):2011.
 Zhou Xu,Quan Zhengxue,Luo Xiaoji,et al.Role of Id1 gene in maintenance of cartilage phenotype in BMP-2-induced intervertebral disc cells[J].J Third Mil Med Univ,2015,37(10):2011.
[2]李建明,初同伟,周跃.BMP-2和bFGF在强直性脊柱炎活动期骶髂关节滑膜组织中的表达[J].第三军医大学学报,2008,30(03):251.
 LI Jian-ming,CHU Tong-wei,ZHOU Yue.Expressions of BMP-2 and bFGF in synovial tissues of cacroiliac joint in the patients with active ankylosing spondylitis[J].J Third Mil Med Univ,2008,30(10):251.
[3]周年,黄伟,廖军义,等.低氧诱导因子-1α对骨形态发生蛋白2诱导的干细胞成软骨、成骨分化的影响[J].第三军医大学学报,2014,36(12):1243.
 Zhou Nian,Huang Wei,Liao Junyi,et al.HIF-1α potentiates BMP2-induced chondrogenic differentiation but inhibits osteogenic differentiation in stem cells[J].J Third Mil Med Univ,2014,36(10):1243.
[4]杨物鹏,许建中.BMP-2和TGFβ1对培养兔关节软骨细胞代谢的影响[J].第三军医大学学报,2000,22(12):0.[doi:10.16016/j.1000-5404.2000.12.007 ]
 YANG Wu peng,XU Jian zhong.[J].J Third Mil Med Univ,2000,22(10):0.[doi:10.16016/j.1000-5404.2000.12.007 ]

更新日期/Last Update: 2018-05-30