Sustained Neurotrophin Release from Protein Nanoparticles Mediated by Matrix Metalloproteinases Induces the Alignment and Differentiation of Nerve Cells
Sustained Neurotrophin Release from Protein Nanoparticles Mediated by Matrix Metalloproteinases Induces the Alignment and Differentiation of Nerve Cells
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DOI：
10.3390/biom9100510
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发表时间：
2019-10-01
期刊：
影响因子：
5.5
通讯作者：
Mori, Hajime
中科院分区：
文献类型：
--
作者：
Matsuzaki, Yuka;Maruta, Rina;Mori, Hajime
细胞因子的时空可获得性，以及由此产生的微环境，对组织发育和动态平衡至关重要。使用可溶蛋白质在体外创建浓度梯度是具有挑战性的，因为它们不能提供自我维持的来源。为了模拟体内细胞外基质(ECM)持续分泌细胞因子的过程，我们将一种货运蛋白包裹到昆虫病毒衍生的蛋白中形成纳米共晶体，并研究了基质金属蛋白酶-2(MMP2)和基质金属蛋白酶-8(MMP8)介导的这种货运蛋白的释放。具体地说，当神经营养因子神经生长因子(NGF)被包裹到纳米颗粒中时，其释放受到PC12神经细胞株分泌的MMPs的促进。当这些NGF纳米颗粒被发现在盖玻片上以创建一个均匀的圆形磁场时，可以观察到PC12细胞通过其延伸的轴突沿着NGF纳米颗粒磁场的外围移动和排列。通过tau、神经丝和GAP-43特异性标志物的表达证实神经细胞的分化。延长的轴突和生长锥体之间也有联系，连接蛋白43的表达与缝隙连接的形成是一致的。生长锥之间有非常细小的丝状孢子伸展和连接。我们的研究表明，晶体蛋白纳米颗粒可以用来产生高度稳定的细胞因子梯度微环境，调节神经细胞的排列和分化。这项技术极大地简化了蛋白质浓度梯度的创建，并可能导致治疗神经元损伤和疾病。
The spatial and temporal availability of cytokines, and the microenvironments this creates, is critical to tissue development and homeostasis. Creating concentration gradients in vitro using soluble proteins is challenging as they do not provide a self-sustainable source. To mimic the sustained cytokine secretion seen in vivo from the extracellular matrix (ECM), we encapsulated a cargo protein into insect virus-derived proteins to form nanoparticle co-crystals and studied the release of this cargo protein mediated by matrix metalloproteinase-2 (MMP-2) and MMP-8. Specifically, when nerve growth factor (NGF), a neurotrophin, was encapsulated into nanoparticles, its release was promoted by MMPs secreted by a PC12 neuronal cell line. When these NGF nanoparticles were spotted onto a cover slip to create a uniform circular field, movement and alignment of PC12 cells via their extended axons along the periphery of the NGF nanoparticle field was observed. Neural cell differentiation was confirmed by the expression of specific markers of tau, neurofilament, and GAP-43. Connections between the extended axons and the growth cones were also observed, and expression of connexin 43 was consistent with the formation of gap junctions. Extensions and connection of very fine filopodia occurred between growth cones. Our studies indicate that crystalline protein nanoparticles can be utilized to generate a highly stable cytokine gradient microenvironment that regulates the alignment and differentiation of nerve cells. This technique greatly simplifies the creation of protein concentration gradients and may lead to therapies for neuronal injuries and disease.