Rspo2-LGR通路在胚胎干细胞向血管平滑肌细胞分化和血管损伤修复中的作用及机制研究

Embryonic stem cells (ESCs) possess capacity of indefinite self-renewal and potential for multi-lineage differentiation. ESCs can be induced into vascular smooth muscle cell (VSMC) in vitro, but the underlying mechanism of differentiate remain unknown. R-spondin (Rspo) family have been found to be a group of secreted proteins essential for development, and Rspo receptors LGRs are important epithelial stem cell markers. By applying our well-establish VSMC differentiation model, we have recently found that Rspo2 is critical during SMC differentiation from mouse embryonic stem (ES) cells. In this study, we resolve to 1) determine the role of Rspo2-LGR pathway in VSMCs differentiation from ESCs, through manipulating Rspo2-LGR signaling and observing corresponding change in expression of VSMCs marker and transcription factor, and function of differentiated VSMC; 2) determine if Wnt/β-catenin signaling is required to mediate Rspo2-induced VSMCs differentiation, through observing the effect on VSMCs differentiation by manipulating Wnt/β-catenin signaling; 3) determine whether Rspo2 exert a protective role in neointimal formation in response to vascular injury. Results of our study would help to better understand the biological mechanisms underlying VSMC differentiation and injury-induced neointima formation, and could provide potential drug target therapy for treating vascular disease, and lay a solid foundation for stem cell application in vascular regenerative medicine.

胚胎干细胞(ESCs)具有无限增殖和多向分化的潜能，可在体外诱导为血管平滑肌细胞(VSMCs)，但分化机制尚不明确。R-spondin(Rspo)家族是新发现的与发育密切相关的分泌性蛋白，其受体LGR是重要的上皮干细胞分子标记物。本课题组在前期研究中发现Rspo2对小鼠ESCs向VSMCs分化过程起关键作用。本研究拟：1)通过增强或抑制分化过程中Rspo2-LGR通路，明确其对小鼠ESCs向VSMCs分化的调控作用；2)通过激活或抑制分化过程中Wnt/β-catenin通路，探讨Rspo2的促ESCs向VSMCs分化作用是否由Wnt/β-catenin通路介导；3) 在小鼠血管损伤模型中验证Rspo2是否在血管损伤后新生内膜形成中起保护作用。本研究有助于深化对VSMCs分化机制的认识，为今后有效地防治血管疾病提供新的药物靶点，并对干细胞在血管再生医学的应用奠定基础。

众多研究表明，干细胞或许通过分化为血管平滑肌细胞参与包括动脉粥样硬化在内的血管损伤性疾病，然而机制尚不明确。Rspo2是新发现的、与骨骼肌发育密切相关的分泌性蛋白，其在干细胞分化血管平滑肌细胞中的作用尚未有任何报道。本项目研究发现，Rspo2表达量在胚胎干细胞向血管平滑肌细胞分化的过程中随着时间的延长mRNA表达量增加，与平滑肌标志物SmαA、SM22、SM-MHC变化趋势一致。用重组蛋白和质粒分别过表达Rspo2后，发现平滑肌标志物表达量增高而抑制Rspo2表达能够降低平滑肌标志物的表达。体外动物实验进一步证明Rspo2促进胚胎干细胞向血管平滑肌细胞分化的作用。进一步，通过q-PCR和Western Blot实验，我们证明Rspo2是通过作用于细胞表面受体LGR5而不是LGR4/6起作用的；同时，wnt3a-β-catenin通路也参与了这一过程。最后，我们利用q-PCR、双荧光素酶基因报告实验、凝胶迁移实验（EMSA）、染色质共沉淀（Chip）实验充分证明了Rspo2能够促进平滑肌分化关键转录因子SRF表达量增加并结合于平滑肌分化标志物SM22的启动子作用元件，发挥作用。从转录水平，揭示了Rspo2的作用机制。综上所述，我们首次证明了Rspo2能够促进胚胎干细胞向血管平滑肌细胞分化，这种作用是通过结合其受体LGR5和wnt3a-β-catenin信号通路、推动SRF结合于平滑肌标志基因的启动子上起作用的。