CN1307199C - 骨形态发生蛋白2活性肽及制备方法和应用 - Google Patents
骨形态发生蛋白2活性肽及制备方法和应用 Download PDFInfo
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Abstract
一种骨形态发生蛋白2活性肽,其特征在于其结构为:S[PO4]KIPKASSVPTELSAISTLYLDDD或CCCCDDDS[PO4]KIPKASSVPTELSAISTLYL。它克服了现有BMP-2半衰期短,难以持续的发挥作用以及生产设备、制备工艺非常复杂,生产周期长,产率低,价格亦非常昂贵,难以大规模生产等不足。具有活性位点能充分暴露、其异位成骨能力较强;较易大规模合成,费用更低,稳定性更好,持续时间长等优点。本发明还同时公开了这种骨形态发生蛋白2活性肽的制备方法和应用。
Description
技术领域
本发明属于临床医学领域,更具体地说它涉及一种骨形态发生蛋白2活性肽及这种活性肽的制备方法和在医学上促进骨再生、骨缺损修复上的应用。
背景技术
我国每年因创伤和疾病所致骨缺损患者达数百万,急需大量骨修复材料。近年来,用组织工程学技术,即用人工细胞外基质材料、生长因子与种子细胞复合移植或单独植入修复骨缺损,引起各国高度重视。但目前国内外骨相关研究尚处于起步阶段,有许多问题亟待解决,其中如何研制出具有显著修复骨缺损效果的药物或材料是亟待解决的关键问题之一。
骨形态发生蛋白(Bone morphogenetic proteins,简称BMPs)是目前唯一能诱导异位成骨的一组细胞因子,迄今已发现了大约15种BMP。目前广泛认为BMP-2是BMP家族中最重要的生长因子, BMP-2诱导骨髓间充质干细胞向成骨方向定向分化的能力最强,但其缺点是半衰期短,难以持续的发挥作用。如何大规模的生产BMP-2从而使其在临床上更加广泛地发挥作用一直是骨组织工程研究的一个难点。目前国内外多采用基因工程技术生产BMP-2,但由于其生产设备、制备工艺非常复杂,生产周期长,产率低,价格亦非常昂贵,难以大规模生产,同时亦存在基因工程产品安全性的问题(Wozney J,Seeherman H.Protein-based tissue engineering in bone andcartilage repair.Curr Opin Biotechnol,2004,15(5):392-398.)。另外,大分子蛋白质吸附在材料表面后会随机折迭,活性位点暴露不充分导致其生物活性不高。另一种方法是运用转基因技术将BMP-2基因转入骨髓间充质干细胞(Bone marrow-derived mesenchymal stem cclls,简称BMSCs),通过转基因细胞表达BMP-2,然而亦存在转染效率低、表达时间较短及病毒载体的潜在致癌性等缺点(Chadderdon R,Shimer A,Gilbertson L.Advances in gene therapy forintervertebral disc degeneration[J].Spine J.2004,4(6 Suppl):341S-34.)。因此,以BMP-2为基础的基因治疗还远远不能运用于临床。
发明内容
本发明的目的在于克服上述现有技术的不足之处,而提供一种骨形态发生蛋白2活性肽。
本发明的另一个目的是提供一种制备上述骨形态发生蛋白2活性肽的方法。
本发明的再一个目的是将本发明骨形态发生蛋白2活性肽在临床上应用于促进骨再生、骨缺损的修复。
本发明的目的是通过如下措施来达到:
一种骨形态发生蛋白2活性肽,其结构为:S[PO4]KIPKASSVPTELSAISTLYLDDD(骨形态发生蛋白2活性肽1)或CCCCDDDS[PO4]KIPKASSVPTELSAISTLYL(骨形态发生蛋白2活性多肽2)。
制备上述骨形态发生蛋白2活性肽的方法为:
BMP-2氨基酸序列中的BMP-2II型受体具有很多抗原表位,其特征性的“指节抗原表位”(Kirsch T,Sebald W,Dreyer MK.Crystal structure of the BMP-2-BRIA ectodomain complex.Nat Struct Biol,2000,7(6):492-6.)中具有诱导成骨的核心功能区,其核心功能区中氨基酸序列为(KIPKASSVPTELSAISTLYL)(Saitoa A,Suzuki Y,Ogataa S,et al.Activation ofosteo-progenitor cells by a novel synthetic peptide derived from the bone morphogeneticprotein-2 knuckle epitope.Biochimica et Biophysica Acta,2003,1651:60-67.)
在其一端加上一个磷酸化的丝氨酸、另外一端加上三个天冬氨酸,形成的骨形态发生蛋白2活性肽1(S[PO4]KIPKASSVPTELSAISTLYLDDD);
或在其一端加上四个半胱氨酸、三个天冬氨酸和一个磷酸化的丝氨酸,形成的骨形态发生蛋白2活性肽2(CCCCDDDS[PO4]KIPKASSVPTELSAISTLYL)。
两条多肽均通过常规的FMOC/tBu固相多肽合成法(Barany G,Merrifield RB.1979.Solidphase peptide synthesis.In:Gross E,Meienhofer J.eds.The peptides uo12.New YorkAcademiPc ress.pp 1-284.):采用对碱不稳定的9-芴甲基羰基(Fmoc)保护氨基酸的α-氨基,采用对酸不稳定的叔丁基型或其它保护基团保护氨基酸的侧链官能团,用聚酰胺树脂作为多肽合成的固相载体。所得粗肽经过凝胶层析初步纯化,最后经过高效液相色谱法分析其纯度,冷冻而得。
这两条多肽能够极好的模拟天然骨基质的促发及指导生物矿化的功能,在局部形成偏酸的环境,促进局部的钙、磷自组装沉积到体内局部组织中胶原纤维表面,生长成按同一方向排列的、坚硬的羟基磷灰石微型晶体结构,形成与天然骨极为相似的结构,从而发挥与天然BMP-2类似的功能。
与常规的骨形态发生蛋白药品相比,该活性多肽具有如下的优点:(1)该活性多肽可发挥与其蛋白质类似的作用,同时短链多肽活性位点能充分暴露并与细胞表面相应的受体结合,达到其较好生物活性,实验结果表明其异位成骨能力较强(参见说明书中动物体内异位成骨的实验研究);(2)此活性多肽两端的序列中含有磷酸化的丝氨酸、半胱氨酸(两种氨基酸为极性中性氨基酸)及天冬氨酸(它为酸性氨基酸),多肽在与相关基质材料结合后,在模拟体液中其两端的这些氨基酸能在材料表面形成磷酸基、羧基等阴离子活性基团。这些阴离子基团是体内促发并指导矿化的重要的功能位点,它们对钙磷离子具有很强的的亲和力,能促进钙磷离子的沉积、晶体的成核生长和自组装矿化,起到调控机体内生物自组装矿化的作用。这几种中性及酸性氨基酸的存在使此活性多肽具有了更好的骨诱导活性,促进了其成骨的效能。我们的研究表明磷酸基等阴离子活性基团能更好的促进基质材料的矿化(参见说明书中材料生物矿化方面的实验研究);(3)多肽较易大规模合成,费用更低,大大减轻了骨缺损病人的经济负担;(4)多肽在与相关基质材料复合过程中稳定性更好,且复合后随着材料本体的逐渐降解而释放,持续时间更长,达到了缓释的效果,将更好更快的促进骨缺损的修复。
附图说明
图1为磷酸化丝氨酸的化学结构图。
图2为骨形态发生蛋白2活性多肽1在Wistar大鼠体内异位成骨的CT照片图。图3为骨形态发生蛋白2活性多肽2在Wistar大鼠体内异位成骨的CT照片图。
图4为光镜下(×10倍)8周时骨形态发生蛋白2活性多肽1异位成骨的HE染色切片图。
图5为光镜下(×20倍)8周时骨形态发生蛋白2活性多肽1异位成骨的HE染色切片图。
图6为光镜下(×10倍)8周时骨形态发生蛋白2活性多肽2异位成骨的HE染色切片图。
图7为光镜下(×10倍)8周时骨形态发生蛋白2活性多肽2异位成骨的HE染色切片图。
图8为光镜下(×10倍)8周时置入单纯胶原海绵材料部位的HE染色切片图。
图9为钙离子吸附质量分析结果图。
图10为扫描电镜显示的PLGA-(PEG-ASP)n支架材料(×50倍)图。
图11为扫描电镜显示的第8天时PLGA-(PEG-ASP)n支架材料在模拟体液中(×2000倍)的图。
图12为扫描电镜显示的第16天时PLGA-(PEG-ASP)n支架材料在模拟体液中(×3000倍)的图。
图13为扫描电镜显示的第16天时PLGA-PEG支架材料在模拟体液中(×3000倍)的图。
图14为PLGA-(PEG-ASP)n材料及PLGA-PEG支架材料在各个时间点质量检测结果图。
具体实施方式
下面结合附图详细说明本发明的实施情况:其中图2,3,4,5,6,7,8为动物实验的结果,即骨形态发生蛋白2活性肽1,2置入Wistar大鼠体内后异位成骨的影像学及组织学检验图。其中图9,10,11,12,13为基质材料材料生物矿化的实验研究的结果图。
(1)动物体内异位成骨的实验研究:
①材料的准备:
通过FMOC/tBu固相多肽合成法(现有常规方法)分别合成骨形态发生蛋白2活性肽1(S[PO1]KIPKASSVPTELSAISTLYLDDD)及骨形态发生蛋白2活性肽2(CCCCDDDS[PO4]KIPKASSVPTELSAISTLYL)。
所用的胶原海绵为武汉博士德公司产品。
以5×5×5mm3大小的胶原海绵为支架材料,将两种骨形态发生蛋白2活性多肽以生理盐水溶解,按照0.4mg剂量分别滴加于胶原海绵上,同时将等量的生理盐水滴加于对照的单纯胶原海绵上,待其充分吸收后冻干备用。
②分组:将24只Wistar大鼠随机平均分为两个实验组,即0.4mg骨形态发生蛋白2活性多肽1/胶原海绵组、0.4mg骨形态发生蛋白2活性多肽2/胶原海绵组,对照采用单纯胶原海绵。
③植入多肽/胶原海绵及单纯胶原海绵:
实验大鼠采用氯胺酮腹腔注射麻醉。在背部骶棘肌切口1cm,两个实验组分别植入0.4mg骨形态发生蛋白2活性多肽1/胶原海绵、0.4mg骨形态发生蛋白2活性多肽2/胶原海绵于一侧骶棘肌肌肉间隙内,对侧骶棘肌肌肉间隙内植入同样大小的单纯的胶原海绵。两组小鼠分别在植入后3、6、8周行CT摄片检查,然后处死4只,并对植入材料的局部组织进行取材,甲醛固定后,经石蜡包埋切片,HE染色,进行组织学观察。
实验结果中影像学CT检查显示分别置入了骨形态发生蛋白2活性多肽1、2的动物体内均有明显的异位成骨(图2、图3);组织学检验(图4、图5)显示骨形态发生蛋白2活性多肽1/胶原海绵组可见明显新骨形成,且发育很充分,多数新骨连续存在。组织学检验(图6、图7)显示骨形态发生蛋白2活性多肽2/胶原海绵组可见明显新骨形成,骨小梁较为宽粗,其表面可见成排的活跃的骨细胞。单纯胶原海绵材料组仅见纤维组织形成,未见类骨质形成,到8周时已被完全降解吸收(图8)。
实验结果说明骨形态发生蛋白2活性多肽1及骨形态发生蛋白2活性多肽2均具有良好的异位成骨能力,和骨形态发生蛋白具有类似的成骨活性,在骨组织工程领域具有前景广阔的应用价值。
(2)基质材料材料生物矿化的实验研究
①材料的准备:
高分子材料丙交酯/乙交酯/天冬氨酸-聚乙二醇(英文名称PLGA-(PEG-ASP)n)多元共聚物的合成:由丙交酯(DLLA)、乙交酯(GA)及天冬氨酸-聚乙烯预聚物开环共聚合成。材料制成直径为5mm的圆片状,厚度为2mm。
高分子材料聚(丙交酯-co-乙交酯)-聚乙二醇(英文名称PLGA-PEG)制成直径为5mm的圆片状,厚度为2mm。
②钙离子吸附质量分析:
40片两种多聚物材料共分5组,分别放入含有不同钙离子浓度的NaCl缓冲液(PH=7.4,150mM)的24孔板中,37度条件下放置。各组中钙离子的浓度分别为0.05,0.1,1,5及10mM。48小时后,通过测定溶液中残留的钙离子的量,得出吸附于每份支架材料上的钙离子含量。溶液中的钙离子浓度运用比色分析法测定。
③生物矿化:
将PLGA-(PEG-ASP)n及PLGA-PEG支架材料放置于24孔组织培养皿中,在各孔中均加入15ml的改良后的模拟体液。分别放置0,4,8,12,16天。每天更换新鲜的模拟体液以保证足够的离子浓度。改良后的模拟体液的组成为:H2O:141mM NaCI,4.0mM KCl,0.5mM MgSO4,1.0mM MgCl2,4.2mM NaHCO3,5.0mM CaCl2,and 2.0mm KH2PO4.合成后的溶液用Tris-HCl溶液进行缓冲至pH=6.8。每份材料在每个培养期处理前及处理后均冻干后进行扫描电镜观察及质量检测。
④实验结果及分析
钙离子吸附质量分析结果表明:PLGA-PEG材料上的钙离子吸附质量明显少于PLGA-(PEG-ASP)n材料上钙离子吸附质量。另外,两种材料上钙离子吸附量均随溶液中的钙离子浓度增加而增加(图9)。
生物矿化结果表明:处理前PLGA-(PEG-ASP)n共聚物支架材料扫描电镜如图(图10)。在不同的培养期,扫描电镜显示在PLGA-(PEG-ASP)n共聚物支架材料内部多孔结构内,充满二氧化碳的低晶状的羟基磷灰石纳米晶体持续的成核和生长。随着培养期的延长,材料内部多孔结构内生物矿物的析出范围明显增大。8天时,许多独立的矿化晶体在材料内部多孔结构内生长(图11)。16天时,出现连续的矿化层,生物矿化晶体显示出薄片状的结构形态(图12)。PLGA-PEG支架材料表面在各个培养期均未见明显的矿物生长(图13)。质量检测结果显示随时间的增加PLGA-(PEG-ASP)n材料的质量有明显的增加,PLGA-PEG支架材料组的质量无明显变化(图14)。
目前普遍采用聚乙二醇(PEG)引发丙交酯和乙交酯共聚来改善PLGA材料的亲水性。然而,形成的嵌段共聚物中缺乏功能基团,难以进一步与生物活性分子复合,材料的细胞亲和性改善不明显,且材料诱导钙磷离子的沉积、晶体的成核生长和自组装矿化的能力均不强。我们在PLGA-PEG嵌段共聚物中引入含活性基团的氨基酸序列,以期改善以往报道的该类共聚物中缺乏功能基团的缺陷。
实验结果表明,经含活性基团的氨基酸序列修饰后,产生了表面化学的差异性,对于多聚物支架材料上钙离子吸附量有明显的影响。天冬氨酸-聚乙二醇预聚物修饰的PLGA共聚物,具有丰富的阴离子基团功能。这些阴离子基团是体内促发并指导矿化的重要的功能位点,它们对钙磷离子具有很强的的亲和力,能促进钙磷离子的沉积、晶体的成核生长和自组装矿化,起到调控机体内生物自组装矿化的作用。
序列表
<110>华中科技大学同济医学院附属协和医院
<120>骨形态发生蛋白2活性肽及制备方法和应用
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Claims (3)
1、一种骨形态发生蛋白2活性肽,其特征在于其结构为:S[PO4]KIPKASSVPTELSAISTLYLDDD或CCCCDDDS[PO4]KIPKASSVPTELSAISTLYL。
2、制备权利要求1所述骨形态发生蛋白2活性肽的方法,它包括如下步骤:BMP-2氨基酸序列BMP-2II型受体具有很多抗原表位,其特征性的“指节抗原表位”中具有诱导成骨的核心功能区,核心功能区中氨基酸序列的结构为:KIPKASSVPTELSAISTLYL;
其特征在于:在所述KIPKASSVPTELSAISTLYL的一端加一个磷酸化的丝氨酸,另一端加三个天冬氨酸,可得到骨形态发生蛋白2活性肽1,其结构为:S[PO4]KIPKASSVPTELSAISTLYLDDD;
或在所述KIPKASSVPTELSAISTLYL的一端加四个半胱氨酸、三个天冬氨酸和一个磷酸化的丝氨酸,可得到骨形态发生蛋白2活性肽2,其结构为:CCCCDDDS[PO4]KIPKASSVPTELSAISTLYL。
3、骨形态发生蛋白2活性肽在药物制备中的应用,所述的骨形态发生蛋白2活性肽为骨形态发生蛋白2活性肽1或骨形态发生蛋白2活性肽2;其特征在于该药物用于治疗促进骨再生、骨缺损修复。
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CN103665142A (zh) * | 2013-11-20 | 2014-03-26 | 北京博恩康生物科技有限公司 | 一种诱导成骨短肽及其制备方法和用途 |
CN103665143A (zh) * | 2013-12-16 | 2014-03-26 | 北京博恩康生物科技有限公司 | 诱导成骨多肽、其制备方法和用途 |
CN105601723B (zh) * | 2015-06-11 | 2019-08-09 | 北京奥斯特赛医学科技有限公司 | 一种煅烧骨靶向结合多肽及其合成方法与应用 |
CN104861076B (zh) * | 2015-06-11 | 2018-06-19 | 北京奥斯特赛医学科技有限公司 | 一种融合多肽及其在促进成骨和血管形成中的应用 |
CN105175501B (zh) * | 2015-06-11 | 2019-05-21 | 北京奥斯特赛医学科技有限公司 | 一种促进骨生成的多肽及其合成方法与应用 |
CN106749606B (zh) * | 2016-12-29 | 2018-06-22 | 广州领晟医疗科技有限公司 | 一种用于修复软骨和/或治疗骨关节炎的肽 |
US11565025B1 (en) | 2017-08-25 | 2023-01-31 | Progenica Therapeutics, Llc | Surface enhanced demineralized bone graft material |
CN108276487B (zh) * | 2017-12-28 | 2020-11-17 | 武汉大学 | 一种可促进成骨并抑制破骨的活性多肽及其应用 |
CN110551201B (zh) * | 2019-08-26 | 2020-04-28 | 杭州彗搏科技有限公司 | 一种新型骨形成蛋白2来源的环肽、制备方法及其应用 |
CN111320682B (zh) * | 2020-02-27 | 2022-07-08 | 广州领晟医疗科技有限公司 | 多肽在制备促进软骨修复和/或治疗骨关节炎药物中的用途 |
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US5618924A (en) * | 1986-07-01 | 1997-04-08 | Genetics Institute, Inc. | BMP-2 products |
EP1006126A2 (en) * | 1998-11-12 | 2000-06-07 | Yoshihiko Nishimura | Osteogenic peptides |
EP1288228A1 (en) * | 2001-08-31 | 2003-03-05 | Yoshihiko Nishimura | BMP-2-derived peptides having osteogenetic activity and osteogenetic accelerator containing the same |
Also Published As
Publication number | Publication date |
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CN1752103A (zh) | 2006-03-29 |
US7754683B2 (en) | 2010-07-13 |
CN101273057A (zh) | 2008-09-24 |
CN101273057B (zh) | 2010-12-08 |
US20090082272A1 (en) | 2009-03-26 |
WO2007048324A1 (fr) | 2007-05-03 |
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