cAMP-Epac介导CXCL12/CXCR4轴调控骨髓间充质干细胞旁分泌并促进皮肤创面修复的机制研究

The paracrine secretion of bone marrow mesenchymal stem cells (BMSCs) is closely related to skin wound repair. However, the molecular mechanism of the paracrine effect of BMSCs in wound repair has less been known, which limits the further improvement of the clinical therapeutic effect of BMSCs. The chemokine CXCL12 and its receptor CXCR4 have been well documented as the key signal axis for maintaining tissue repair, and have important regulatory effects on BMSCs paracrine secretion as well, but the molecular mechanism remains unclear. Epac is a new downstream molecule directly activated by cAMP. Its expression is closely related to the cellular function of BMSCs, and participates in various biological processes such as cell proliferation and paracrine secretion. Our previous study confirmed that up-regulation of Epac expression could improve mitochondrial function and increase cell viability of BMSCs. In the latest study, we employed antibody microarray and whole transcriptome sequencing, and observed that the promotion of BMSCs paracrine secretion by CXCL12 might be related to the activation of cAMP signaling pathway and the enhancement of Epac gene expression. Meanwhile, CXCL12 promoted Epac protein expression in BMSCs. Therefore, it is proposed that a large amount of CXCL12 released after the formation of skin wounds may activate the cAMP-Epac signaling to regulate BMSCs paracrine secretion, and thus promote wound repair. Based on the data of pilot study, this study is to systematically study the mechanisms of cAMP-Epac mediated CXCL12/CXCR4 axis in the regulation of BMSCs paracrine secretion and the promotion of skin wound repair. Firstly, the temporal and spatial expression pattern of Epac in BMSCs in animal wound model will be measured, and the effect of CXCL12/CXCR4 on its expression will be explored. Furthermore, cAMP-Epac mediated CXCL12/CXCR4 axis in the regulation of BMSCs paracrine secretion and the promotion of skin wound repair will be studied by GOF/LOF and agonist/inhibitor intervention technologies. Finally, the molecular mechanism of CXCL12/CXCR4 axis on the paracrine effect of BMSCs will be elucidated and verified using CXCR4 inhibitor, Epac inhibitor and Epac-siRNA both in vitro and in vivo. Through the above research, this study will clarify the role of cAMP-Epac signaling pathway in the BMSCs paracrine secretion in wound repair, and reveal the molecular mechanism that cAMP-Epac may serve as an important downstream molecule of CXCL12/CXCR4 axis, which may provide theoretical basis and new drug targets for the clinical optimization of BMSCs therapeutic effects on skin wound repair.

骨髓间充质干细胞（BMSCs）旁分泌作用与皮肤创面修复密切相关。然而，治疗创面时，BMSCs旁分泌作用的生物学调控机制目前报道尚少，限制了BMSCs临床治疗效果的进一步优化。趋化因子CXCL12及其受体CXCR4是维持组织修复的关键信号轴，并对BMSCs旁分泌有重要调节作用，但调控机制尚不清楚。Epac是cAMP直接激活的下游新效应分子，其表达与BMSCs的细胞功能密切相关，参与细胞增殖和旁分泌等多种生物学过程。申请者前期研究证实，Epac表达上调可以改善BMSCs线粒体功能并提高细胞存活率。进一步预实验联合抗体芯片及全转录组测序结果显示，CXCL12改善BMSCs炎性因子分泌可能与激活cAMP途径和增强下游基因Epac表达有关；同时，在蛋白水平证实CXCL12可以上调BMSCs中Epac表达。因此，提出创面形成后，随之释放的大量CXCL12通过激活cAMP-Epac信号通路，调控BMSCs旁分泌并促进创面修复。本项目拟在前期工作基础上，进一步探讨cAMP-Epac信号通路在CXCL12/CXCR4轴调控BMSCs旁分泌促进创面修复中所发挥的作用和可能的机制。首先观察动物创面模型BMSCs中Epac的时空表达特征，探索CXCL12/CXCR4对其表达的影响；进而通过GOF/LOF、激动剂/抑制剂等干预策略，研究cAMP-Epac介导CXCL12/CXCR4轴调节BMSCs旁分泌促进创面修复的作用；最后借助CXCR4阻断剂、Epac抑制剂和Epac-siRNA，在体内外模型中阐明并验证CXCL12/CXCR4轴对BMSCs旁分泌作用的分子调控机制。通过以上研究，本课题将明确cAMP-Epac信号通路在BMSCs旁分泌治疗创面中的作用，揭示其介导CXCL12/CXCR4轴发挥作用的分子机制，为临床进一步拓展BMSCs应用、优化BMSCs治疗皮肤创面效果提供理论依据和新靶点。

难愈性创面修复是临床治疗难题。骨髓间充质干细胞（BMSCs）具有确切地促进急慢性创面修复的作用，并且此修复潜能主要来自于其旁分泌作用。既往研究表明，趋化因子CXCL12及其受体CXCR4是维持组织修复的关键信号轴，并对BMSCs旁分泌有重要调节作用，但具体调控机制不清。cAMP直接激活的鸟苷酸交换因子（Epac）被活化后，可以通过激活小分子G蛋白酶Rap1参与细胞功能调控。本研究前期联合抗体芯片及全转录组测序分析结果提示，CXCL12通过激活cAMP途径并增强下游基因Epac表达调控BMSCs炎性因子分泌。本研究首先检测正常BMSCs中Epac两种亚型Epac1和Epac2的基因和蛋白表达水平，结果发现BMSCs主要表达Epac1。细胞功能实验结果显示，Epac1激动剂8-pCPT处理BMSCs后，可以明显促进BMSCs细胞增殖和ATP生成。同时，8-pCPT可以明显促进BMSCs细胞迁移。CXCL12处理一定时间后，BMSCs中Epac1表达水平显著升高，并且Erk磷酸化形式明显增加。8-pCPT同样可以明显增加BMSCs中p-Erk表达水平，而Epac1特异性抑制剂ESI-09则能逆转这种效应。通过GST pull-down实验，检测发现CXCL12和8-pCPT均能够明显提高BMSCs中Rap1-GTP/Total Rap1比值。进一步通过细胞因子矩阵检测发现，8-pCPT明显提高BMSCs的VEGF分泌水平。通过构建小鼠背部全层皮肤创面模型，实验结果显示移植8-pCPT 处理后的BMSCs组小鼠创面愈合时间明显缩短，皮肤再上皮化更加明显，胶原沉积更有序。本研究通过体内外实验探索了CXCL12/CXCR4轴与Epac调控应答对BMSCs旁分泌与功能的影响及分子机制，并在体内进行了生物疗效的验证，为临床提高BMSCs促进皮肤创面修复潜能提供了新的潜在干预靶标和理论基础。