Quantification of release and activity of the signaling factors BMP-2, TGF-ß3, Smad8 L+MH2

信号因子 BMP-2、TGF-α3、Smad8 L MH2 的释放和活性定量

• 批准号: 270206734
• 负责人: Professorin Dr. Heike Bunjes
• 金额: --
• 依托单位: Institut für Pharmazeutische Technologie
• 依托单位国家: 德国
• 项目类别: Research Units
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/270206734?language=en
• 关键词: Quantification; release; activity; signaling; factors

Project 6 is concerned with the demonstration of a successful spatial gradation of the growth factors previously incorporated into the implant in order to induce cell differentiation and of the temporal control of their release. Moreover, the preservation of protein activity during processing and release is going to be investigated. For this purpose, suitable methods are established that allow the determination of amount and activity of the growth factors in relevant media. For a rapid and cost-efficient semiquantitative determination of the amount of released proteins as well as for a control for their molecular integrity (in particular, with regard to degradation), protein electrophoresis (denaturing and also native) in combination with Western Blots is employed. In particular with regard to visualization investigations with fluorescence detection will be carried out. Advanced quantitative analysis of the amount of released protein will be performed by enzyme-linked immunosorbent assay (ELISA) and Selected Reaction Monitoring which is going to be established as an alternative quantification method. Reporter tests (promotor elements of target genes of the TGF-β/BMP signaling pathway) and differentation analysis (gene expression) of human mesenchymal stem cells allow conclusions on the biological activity of the proteins. Further, it will be investigated how far storage and transport or the use of additives in release media may influence results and whether there are cross-reactions between the target proteins. Experimental setups and suitable media for release studies on the graded implants and their components will be developed and optimized with respect to their suitability regarding quality control as well as a later performance in vivo. In addition to the use of liquid release media hydrogels are going to be used as acceptor systems for protein release. In this context, the use of microdialysis and/or open flow microperfusion for the collection of released growth factors is going to be evaluated and special release cells will be designed. These methods will be used to investigate the release of growth factors from graded implants and their components obtained from P5 and the activity of the released proteins. Based on the results from these experiments, the release systems will be further developed. Together with P5 the potential of using specifically prepared aggregates of BMP-2 as a depot on the implants will be evaluated. In the second funding period, there will be a special focus on the establishment of an in vitro-in vivo-correlation (IVIVC). Moreover, in a combined effort with the other projects, P6 will elaborate concepts for the production of a sterile implant.

项目6涉及成功地展示先前结合到植入物中的生长因子的空间渐变，以便诱导细胞分化和对其释放的时间控制。此外，还将对蛋白质在加工和释放过程中的活性保存进行研究。为此，建立了适当的方法，允许确定相关介质中生长因子的数量和活性。为了快速和经济有效地半定量测定蛋白质的释放量以及控制其分子完整性(特别是关于降解)，采用蛋白质电泳法(变性的和天然的)结合Western blotts。特别是在可视化方面，将利用荧光检测进行调查。蛋白质释放量的高级定量分析将通过酶联免疫吸附试验(ELISA)和选择性反应监测进行，这将作为一种替代的定量方法。报告测试(转化生长因子-β/骨形态发生蛋白信号通路靶基因的启动子元件)和人骨髓间充质干细胞的分化分析(基因表达)可以得出蛋白质生物活性的结论。此外，还将调查在多大程度上储存和运输或在释放介质中使用添加剂会对结果产生影响，以及目标蛋白之间是否存在交叉反应。将开发和优化实验装置和合适的介质，以进行分级植入物及其组件的释放研究，以确保它们在质量控制方面的适用性以及以后的体内表现。除了使用液体释放介质外，水凝胶还将被用作蛋白质释放的受体系统。在这种情况下，将对微透析和/或开放流动微灌流用于收集释放的生长因子进行评估，并将设计特殊的释放池。这些方法将被用来研究从P5获得的分级植入物及其成分的生长因子的释放以及释放的蛋白质的活性。在这些实验结果的基础上，将进一步开发释放系统。与P5一起，将评估使用专门准备的BMP-2聚集体作为植入物仓库的潜力。在第二个资助期，将特别注重建立体外-体内-相关性(IVIVC)。此外，在与其他项目的联合努力中，P6将详细阐述生产无菌植入物的概念。