功能性仿生基底膜的体外构建及其促进表皮干细胞增殖的分子机理研究

It has been well-known that the basement membrane (BM) is the key factor affecting the quality of wound repair as well as a big challenge in the construction of tissue-engineered skin. Our research group had previously fabricated a type of novel micro-nano-micro structured nanofibrous scaffold which could realize the isolation between skin keratinocytes (KCs) and fibroblasts (Fbs), also raised the expression level of integrin β1, Rac1 and Cdc42 of KCs. Due to the crucial role of Integrin β1 in regulating the adhesion and proliferation of ESCs, with the established evidence, we proposed the hypothesis that biomimetic BM fabricated by our research group could promote the adhesion and proliferation of ESCs (epidermal stem cells) through activating integrin signaling pathway to maintain stem cell properties. So our team plans to adopt the method of physicochemical and biological modification to fabricate a new type of the bionic basement membrane with incorporated bioactive molecules, its biological character will be studied and its prognosis in the body will be investigated. Meanwhile, western-blot and RT-PCR methods will be used to detect the basement membrane regulating the expression level of integrinβ1 in the ESCs. Finally, it will be clarified the molecular mechanism that the bionic BM regulates ESCs and further promote the development of tissue-engineered BM substitute research.

基底膜是影响创面修复质量的重要因素也是组织工程皮肤面临的难点问题。项目组构建了具有特殊微-纳-微结构的仿生纳米纤维支架可实现皮肤角质形成细胞（KCs）与成纤维细胞（Fbs）的有效隔离并活化KCs的整合素β1（Integrin β1）、上调Rac1与Cdc42表达水平。由于Integrin β1对基底表皮干细胞（epidermal stem cells）在基底膜上的粘附与增殖起关键调控作用，我们认为通过改性制备的仿生基底膜介导的integrin水平上调可促进ESCs的粘附与增殖而维持其“干”性。因此课题组拟采用理化与生物学改性的方法构建新型仿生基底膜并研究其生物学性状与在体功能；采用Western-blot与RT-PCR方法检测基底膜对ESCs的关键调控分子integrinβ1表达的影响。最终阐明仿生基底膜调控ESCs增殖的分子调控机制，并有望促进组织工程基底膜替代物的发展。

实现基底膜结构与功能重建成为限制组织工程皮肤发展的关键问题。项目组采用理化与生物学改性方法制备复合生物活性分子的新型纤维支架以高度模拟天然皮肤基底膜微环境，研究该支架的生物学特性与在体构建情况；并验证该仿生基底膜对ESCs粘附、增殖与干细胞特异性标记物表达的影响，在此基础上筛选与其增殖、分化、凋亡以及细胞形态调控相关的关键蛋白分子及相关分子信号通路。最终验证仿生基底膜-ESCs复合物对创面愈合作用效果，并证实与仿生基底膜支架结合ESCs促进创面愈合相关的功能基因群。研究结果表明：（1）采用静电纺丝混纺法制备具有微-纳-微三层结构的聚己内酯/醋酸纤维素/壳聚糖复合纳米纤维支架可模拟天然基底膜结构；并在实现Kcs与Fbs的有效隔离的同时允许其信号交流；此外制备的仿生支架可显著促进基底膜成分Laminin-5表达；（3）制备仿生支架可显著促进创面表皮化，表真皮层间连接紧密；与对照组相比，Kcs-仿生基底膜-Fbs复合物覆盖组处理的创面组织活化整合素β1与磷酸化FAK水平显著提高；（4）与对照组相比，ESCs在支架上相互连接，形成集落状。细胞伸展面积更小，EFF更高，表皮干细胞进入细胞周期的细胞数量较少，细胞增殖的速率较慢，且表皮干细胞的分化显著被抑制，表明仿生基底膜可模拟干细胞龛环境，使表皮干细胞处于低分化状态；（5）在支架提供的微环境中，表皮干细胞蛋白表达谱有显著差异。整合素家族蛋白和核糖体、细胞代谢途径相关蛋白可能与支架对表皮干细胞生物学行为调控相关；（6）纳米纤维支架-ESCs膜片能够显著促进创面的愈合；新生皮肤胶原排布更加整齐、基底膜更加完整；新生皮肤基底部表皮干细胞特异性标志物表达强度更高，干细胞活性更强。本课题研究成果不仅为构建完全替代天然基底膜的新型材料提供了理论基础与技术支撑，而且通过阐明仿生基底膜调控ESCs增殖的分子机制，为材料诱导组织再生提供了科学依据。