LBL改性PCL-Cellulose纳米支架激活Kc细胞的Integrin-FAK信号通路机制研究

An insightful understanding of the mechanism involving the effects of nanofibrous matrix on the adhesion, proliferation, and migration of(keratinocytes, Kc) is important for exploring the scaffold/cell interaction, our previous study showed that LBL modified electrospun polycaprolactone (PCL)-based nanofibrous matrices with different topographical feature and physicochemical characteristics could significantly affect the biological behavior of Kc attached, besides, the nanofibers could significantly activated integrinβ1 and promoted the expression of FAK(focal adhesion kinase) (Tyr 397), however, the underlying regulatory mechanism and biological effect of Kc cells remained unclear. Based on these findings, we plan to further confirm the effect of nanofibrous scaffold on the wound healing via in vivo model; and explore the regulatory effect of nanofibrous matrix with various topographical features (fiber diameter, pore size and fiber organization) and surface chemistry on the adhesion, proliferation, and migration of Kc cells, furthermore, we aimed to determine the regulatory involvement of integrin β1 and FAK in promoting the wound healing on nanofibrous matrices, this project wound be helpful to further elucidate the regulatory mechanisms of scaffold/cell interaction.

深入研究纳米纤维材料对于细胞的增殖与迁移的作用机制对于了解材料与细胞间的相互作用具有重要意义。项目组前期工作显示：改性聚己内酯基纳米纤维支架的拓扑结构与理化特性可显著影响角质形成细胞(keratinocytes, Kc)的生物学行为，此外，支架可激活Kc细胞Integrinβ1并促进细胞FAK(Tyr-397)磷酸化水平上调，但是纳米支架是否通过Integrinβ1激活FAK信号通路从而影响细胞的生物学行为还有待证实。因此，本项目在前期研究基础上，拟进一步探究支架的拓扑结构（纤维直径、孔径与取向性）与表面特性对于Kc细胞粘附、增殖与迁移的调控作用，阐明支架介导的integrinβ1与FAK分子在促进创面愈合中发挥作用的具体分子机制，本项目的开展有助于进一步阐明支架-细胞之间的相互作用分子机制。

深入研究纳米纤维基质对于细胞的增殖与迁移的作用机制对于了解材料与细胞间的相互作用具有重要意义。本项目在探究支架的拓扑结构（纤维直径、孔径与取向性）与表面特性对于Kc细胞粘附、增殖与迁移的调控作用的基础上，进一步阐明支架介导的integrinβ1与FAK分子在促进创面愈合中发挥作用的具体分子机制。.项目组研究结果表明：改性聚己内酯-胶原复合纳米纤维支架的纤维形貌、取向性与复合的生物活性分子可显著影响角质形成细胞(keratinocytes, Kc)的生物学行为，扫描电镜、细胞示踪、F-actin染色与共聚焦显微镜观察结果显示具有较细纤维直径的聚己内酯-胶原复合纳米纤维可显著促进Kcs的粘附与增殖。且纳米纤维支架具有良好的体内生物相容性、可降解性，可促进细胞外基质蛋白（ECM）沉积并可显著加速创面愈合。此外，免疫荧染色与Western Blotting结果表明支架可促进Kc细胞内活化整合素β1（active Integrinβ1）与磷酸化黏着斑激酶（FAK(Tyr-397)）表达水平上调。在此基础上，课题组采用CRISPR-Cas9体系成功构建条件性integrinβ1基因敲除细胞系，纤维支架与细胞共培养后的RT-PCR与Western Blotting实验表明：与条敲组无integrinβ1表达相比，纳米支架共培养组Kcs内可检测到active Integrinβ1与Tyr397 FAK高表达，该结果提示制备的纳米纤维支架可通过介导Integrinβ1激活FAK信号通路从而影响细胞的生物学行为，并加速创面表皮化。.本项目的开展有助于进一步阐明支架-细胞之间的相互作用分子机制，并为构建功能化表皮细胞膜片及其在创面愈合领域的应用提供了科学依据与可借鉴的新模式。