BMP-2的共价结合利用表面上的自组装单层膜法
Summary
我们描述了用于实现BMP-2的有效固定化在表面上的方法。我们的方法是基于一个自组装单层实现共价通过其游离胺残基的BMP-2的结合的形成。这个方法是研究信令在细胞膜的有用工具。
Abstract
骨形态发生蛋白-2（BMP-2）是嵌入在骨组织中的细胞外基质的生长因子。 BMP-2作为间充质细胞分化为成骨细胞的触发器，从而刺激愈合和新生骨形成。与支架结合临床使用的重组人BMP-2（的rhBMP-2）提出了一些争议，根据演示文稿的方式和交付量。这里介绍的协议提供了一种简单而有效的方式提供的BMP-2的体外研究细胞。我们描述了如何形成自组装单分子层组成的异双功能连接物，并显示在下面的结合步骤中获得的rhBMP-2的共价固定。用这种方法，可以实现BMP-2的持续文稿，同时保持了蛋白质的生物活性。事实上，BMP-2的表面固定允许有针对性的调查，防止非特异性的广告orption，同时减少的生长因子的量和，最值得注意的是，阻碍了从表面不受控制的释放。由BMP-2引起的短期和长期的信号传导事件正在发生时，细胞暴露于表面呈共价固定的rhBMP-2，使得该方法适于在体外研究细胞对BMP-2刺激。
Introduction
骨形态发生蛋白-2（BMP-2）是转化生长因子（TGF-β）家族，并作为新生骨形成的诱导剂，以及几种组织的胚胎发育和成年人中调节动态平衡1-3的成员。 具有生物活性的同型二聚体的BMP-2蛋白的每个单体包含一个“半胱氨酸结”的主题，它是高度保守的所有骨形成蛋白4。六七个半胱氨酸残基形成稳定的每个单体分子内二硫键，而第七个半胱氨酸参与二聚化，形成了两种单体5,6之间的分子键。这个高度保守的半胱氨酸结定义了BMP-2蛋白的三维结构，并确定其独特的性能，如对热，变性剂和酸性pH 7-9的阻力。 BMP-2的结合的丝氨酸/苏氨酸激酶的跨膜受体，从而诱导信号转导<sup> 10-12。取决于受体寡聚化的模式下，不同的信号通路被激活：一个Smad蛋白非依赖性信号传导级联通过p38信号导致的碱性磷酸酶诱导，而Smad蛋白依赖性途径通过受体磷酸化的结果中的Smad复合物的核转位和活化的转录活化具体靶基因，例如分化的抑制剂（ID）12-14。
在骨，BMP-2诱导间充质干细胞分化为成骨细胞，从而刺激愈合和从头形成骨。目前，重组表达的BMP-2是临床应用，以提高骨折部位的愈合。在骨组织工程常见的策略是利用注射生长因子，相比本地投递系统，是创伤小。然而， 在体内研究和临床应用表明，短的生物半衰期，非特异性禄alization和BMP-2的快速局部间隙可能导致一些地方，异位和系统性问题15。因此，为了获得有效的演示，BMP-2的范围内或之上的材料的包封或固定是必要的，它的本地和持续递送于靶位点。持续释放可以通过非共价的保留的方法，如物理截留，吸附或离子络合16来实现。然而，它是已知的蛋白质的非特异性吸附到表面可导致分子17的变性。为共价的生长因子结合，已经开发了不同类型的支撑件，在过去十年。使用双官能连接基的分子靶向的蛋白质的氨基或羧基基团例如，是一种类型的，这并不一定需要的蛋白质修饰，以实现其固定的方法。事实上，当蛋白质修饰提供调控蛋白定向的优势，引入人工结构域，肽标记和特定于站点的链可改变生长的生物活性因子17。因此，为了规避变性由于与支撑材料的相互作用，表面可以事先官能化，例如，有自组装单分子层的连接分子（SAM），接着是所需的因子18的耦合。我们已经使用了基于SAM的方法，共价固定化的BMP-2在表面上通过靶向其游离胺残基且已经表明，固定化的蛋白保留其短期和长期的生物活性19。这个协议提供了一种简单而有效的方式提供的BMP-2的细胞的体外研究在其上发生在细胞膜和调节负责信令成骨细胞内信号传导的机制。
Protocol
1。 11-Mercaptoundecanoyl-N-琥珀酰亚胺酯（MU-NHS）的合成逐滴加入500毫克N-羟基琥珀酰亚胺的溶液和30mg 4 – （二甲氨基）吡啶在10ml丙酮（PA）的1克11-巯基十一酸的40ml二氯甲烷（PA）在室温（RT）。 将反应冷却至0℃，并逐滴加入1.1克N，N' -二环己基碳在10毫升二氯甲烷中（氮气氛下）。保持在低温下进行1小时反应，然后在室温搅拌过夜。 过滤出沉淀，干燥，?…
Representative Results
在我们的设置中，黄金被选为赋形剂，因为它提供了一种生物，但非特异性化学可调系统。另外，自组装单分子膜的应用嗣继承很多益处：通过自组装膜对金属和形式的单层缺陷少其“头部基团”自发地吸附，而其末端官能基团可以被进一步修饰。因此，他们提供一个平台，在受控而高度适应性的方式20定制界面的属性。 对于BMP-2对金涂覆的表面上的固定化，我们使?…
Discussion
在这个协议中，我们描述了功能化与生物活性的rhBMP-2表面的准备。该方法包括两个步骤：在金表面的双官能连接体的自组装单层（SAM）的1）初步形成，2）共价的rhBMP-2蛋白的固定化。在以前的工作中，我们验证了双官能连接子和生长因子的有效结合，并表明表面固定化的rhBMP-2保持其生物活性19。在固定化的形式呈现的生长因子的生物活性也已显示在其他研究中，表明该蛋白的释放以及随?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢教授太平绅士斯帕茨（生物物理化学系，海德堡大学以及新材料和生物系统学系，马克斯普朗克研究所的智能系统，斯图加特）对他的鼎力支持。从马克斯 – 普朗克协会与德意志研究联合会（DFG SFB/TR79到EAC-A）的财政支持也大大承认。
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| N-hydroxysuccinimide | Sigma-Aldrich | 130672 | |
| 4-(dimethylamino)pyridin | Sigma-Aldrich | 522805 | |
| Acetone | AppliChem | A2282 | |
| 11-mercaptoundecanoic acid | Sigma-Aldrich | 674427 | |
| Dichlormethane | Merck | 106050 | |
| N,N'-dicyclohexylcarbodiimide | Sigma-Aldrich | D80002 | |
| Petroleum benzene | Merck | ||
| Glass coverslips | Carl Roth | M 875 | |
| Ethylacetate | AppliChem | A3550 | |
| Methanol | Carl Roth | 4627 | |
| N,N-dimethylformamide | Carl Roth | T921 | |
| rhBMP-2 | R&D Systems | 355-BM | Carrier-free; expressed in E.coli |
| PBS | PAA | H15-002 | |
| NaCl | Carl Roth | HN00.2 | |
| Poly(dimethyl siloxane) (PDMS) | Dow Corning | ||
| Sylgard 184 silicone elastomer kit | Dow Corning | ||
| Anti-rhBMP-2 | Sigma | B9553 | |
| Goat anti-mouse IgG-HRP | Santa Cruz | sc-2005 | Secondary antibody |
| Ampliflu Red assay | Sigma | 90101 | |
| Dulbecco's Modified Eagle Medium (DMEM) (1x), liquid | Gibco | 41966 | High glucose |
| Fetal Bovine Serum (FBS) | Sigma | F7524 | Sterile filtered, cell culture tested |
| Pen/Strep | Gibco | 15140 | |
| Trypsin 0.05% (1x) with EDTA 4Na | Gibco | 25300 | |
| Glycine (0.1 M) | Riedel-de Haën | 33226 | |
| IGEPAL CA-630 (1%) | Sigma | I8896 | Lysis buffer (ALP assay)19 |
| Magnesium chloride (MgCl2)(1 mM) | Carl Roth | HNO3.2 | |
| Zinc chloride (ZnCl2) (1 mM) | Carl Roth | 3533.1 | |
| p-nitrophenylphosphate (pNPP) | Sigma | S0942 | Phosphatase substrate |
| Anti-mysin heavy chain (MHC) | Developmental Studies Hybridoma Bank, University of Iowa | MF20 | Monoclonal antibody |
| Alexa Fluor 488 Goat anti-mouse IgG | Invitrogen | A11001 | |
| DAPI | Sigma | D9542 | |
| Equipment | |||
| Ultrsonic bath (Sonorex Super RK 102H), Frequency 35 kHz | BANDELIN electronic GmbH & Co. KG | ||
| MED 020 Sputtercoating system | BAL-TEC AG | Coating conditions Cr: 120 mA, 1.3 x 10-2 mbar, 30 sec Au: 60 mA, 5.0 x 10-2 mbar, 45 sec |
|
| Tecan Infinite M200 Plate reader | Tecan |
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Cite This Article
Pohl, T. L. M., Schwab, E. H., Cavalcanti-Adam, E. A. Covalent Binding of BMP-2 on Surfaces Using a Self-assembled Monolayer Approach. J. Vis. Exp. (78), e50842, doi:10.3791/50842 (2013).