力学刺激调控miR-100诱导脂肪干细胞向平滑肌细胞分化的作用和机制

The use of stem cells for repairing the smooth muscle lesions in vascular diseases is becoming the focus of regenerative medicine and tissue engineering. It has been demonstrated by accumulated evidence that mechanical stimuli regulate smooth muscle cells (SMCs) phenotype, functionality and matrix remodeling. Our preliminary studies show that mechanical stimulation significantly promotes SMC differentiation of growth factor-induced adipose-derived stem cells (ASCs) while increases the miR-100 expression remarkably during the process. According to the related literature and our previous research, we supposed that the appropriate mechanical load stimulation can promote ASCs to differentiate into SMCs, while miR-100 is a positive regulatory factor for this process. Through taking mechanical stimulated ASCs as the study object, we construct miR-100 over-expression and antisense expression vector, and finally identify the regulatory role of miR-100 in the process of ASCs differentiated into SMCs in this study. We also construct the smooth muscle tissue in vitro, and observe the ability of miR-100 regulate SMC differentiation in ASCs and forming smooth muscle tissue as well. The results will deeply elucidate the roles and mechanisms of SMC differentiation of ASCs which regulated by mechanical stimulation and miR-100, and also represent a promise strategy for SMC differentiation of stem cells and smooth muscle tissue construction in the future.

利用干细胞进行组织构建来修复平滑肌病变引起的血管疾病等是目前再生医学和组织工程研究的重点。有文献报道，力学刺激可调控平滑肌细胞（SMCs）的表型、功能及基质重塑。课题组前期研究表明：一定的力学刺激可明显促进生长因子诱导的脂肪干细胞（ASCs）向SMCs方向分化，且在分化过程中miR-100表达显著升高。结合文献及前期工作，我们推测：适宜的力学加载刺激可促进ASCs向SMCs的定向诱导分化，而miR-100是调控此分化的正向调节因子。本研究将构建miR-100过表达及反义表达载体，以力学加载刺激的ASCs为研究对象，明确miR-100在ASCs向SMCs分化过程中调控作用；并进行体外平滑肌组织构建，在组织水平观察miR-100调控ASCs成肌分化能力。本研究将深入阐明力学刺激及miR-100调控ASCs定向成肌分化的作用和机制，并有望为干细胞成肌分化和平滑肌组织构建的实施提供新的思路和途径。

获取收缩型、有功能的平滑肌细胞对构建平滑肌组织至关重要，而力学刺激在调控细胞分化、平滑肌细胞表型及基质分泌重塑方面发挥重要作用。本项目主要筛选促进ASCs向SMCs定向诱导分化的最佳力学刺激条件，并确定miR-100是力学刺激介导ASCs向SMCs定向分化的关键调控基因；同时探讨miR-100特定靶基因，并观察靶基因CaMKIIγ调控ASCs向SMCs分化的作用机制；以及观察体外miR-100调控平滑肌组织形成能力。目前已确定促进ASCs向SMCs分化的最佳力学刺激条件（形变量5%、施加频率1Hz、体外机械拉伸刺激为12h）；明确miR-100是最佳力学刺激（5%-1Hz-12h）介导ASCs向SMCs定向诱导分化的关键调控基因，是力学反应特异性miRNA，其可促进平滑肌细胞特异性标记物的表达；根据基因芯片结果验证了miR-100的靶基因为CaMKIIγ，并进一步阐明了CaMKIIγ调控ASCs向SMCs分化的机制，初步明确miR-100/CaMKIIγ通路在调控力学刺激介导ASCs向SMCs分化过程中的作用。项目组在前期研究的基础上，进一步完善了电纺丝技术制备的管状高分子材料，并以此支架，接种诱导分化的平滑肌细胞，进一步优化实验条件来构建纳米载体携载 miRNA 质粒，从而为基因治疗做好铺垫。目前观察体外miR-100调控ASCs分化形成平滑肌组织能力的研究还在进行中。此外，课题组还开展了miR-145在调控ASCs向SMCs分化中的机制研究。另外，我们对脂肪干细胞促进皮瓣血管化及其机制，以及异体来源的脂肪干细胞促进成骨形成并修复骨缺损进行了相关研究，进一步完善了脂肪干细胞的应用领域，以上研究成果发表在“Cytotherapy，Cell Tissue Res，Exp Biol Med和Mol Med Rep”上。