AMI后移植MSCs促进Ly6Chigh单核细胞向Ly6Clow表型转化的机制研究

Our previous studies confirmed that: targeted degradation of CCR2 on monocyte and thereby inhibiting the spleen derived Ly6Chigh monocytes’ rapid mobilization to the infracted myocardium, significantly improved the therapeutic efficacy of transplanted MSCs after AMI; Meanwhile we also found decreased Ly6Clow monocytes migrated into the infarcted myocardium, resulting the absorption and regression of inflammation slowed down significantly. Further experiments reveals that transplanted MSCs can contributes the phenotypic switch of Ly6Chigh monocytes to Ly6Clow and which may related to the expression of Nr4a1, however, the exact potential mechanism is unknown..The project intends further study on the mechanisms that transplanted MSCs regulate Ly6Chigh monocytes to Ly6Clow phenotype: ① Influence and mechanism of TNF-α / LPS on expression of nuclear receptors of Nr4a1 in MSCs; ②Influence and mechanism of Nr4a1 on the secretion of Ang-1/SDF-1 in MSCs ; ③Influence and mechanism of Ang-1/SDF-1 expression on NF-κB of Ly6Chigh monocytes ;.Exploring the above issues will further clarify the exact functional relationship between inflammatory monocytes and MSCs and understand the underlying immunomodulatory mechanism of MSCs on monocytes in addition to paracrine action, so as to provide a new target for preventing cardiac remodeling after AMI

申请者前期研究证实：AMI后特异性降解单核细胞CCR2进而抑制Ly6Chigh细胞快速浸润至梗死心肌，显著改善了MSCs的移植疗效，但发现同期迁移至梗死心肌的Ly6Clow细胞亦明显减少，导致炎症的吸收和消退显著减慢。进一步实验发现移植的MSCs可调节Ly6Chigh细胞向Ly6Clow表型转化，且可能与Nr4a1表达有关，但具体机制不明。.本项目拟进一步研究移植的MSCs调节Ly6Chigh细胞向Ly6Clow表型转化的具体机制：①TNF-α/LPS对MSCs的Nr4a1表达影响及机制分析；②Nr4a1对MSCs分泌Ang-1/SDF-1的影响及机制分析；③Ang-1/SDF-1的表达对Ly6Chigh细胞NF-κB的影响及机制研究；.探讨上述问题将进一步明确MSCs与Ly6Chigh细胞之间的功能关系，理解移植MSCs除旁分泌以外的免疫调节作用机制，为AMI后心肌重塑的治疗提供新靶点。

在既有研究计划和研究进展的指引下，本课题组着重在心肌梗死后炎症单核细胞免疫干预以及改善移植的MSCs存活方面做了一系列工作。第一方面我们着重构建了用于MSCs三维培养的反蛋白石硅纳米结构载体，成功地将骨髓间充质干细胞(MSCs)培养于三维立体反蛋白石硅纳米结构载体结构表面，实现了MSCs的立体可控式生长，研究发现该载体支撑培养下的MSCs的细胞分泌作用更强，抗缺氧及抗凋亡能力更强。将反蛋白石结构的纳米载体荷载的MSCs移植入梗死心肌局部， 14天后的AMI小鼠心肌梗死面积和21天后的AMI小鼠心肌重塑均有明显改善。该部分研究不仅体现了三维反蛋白石纳米载体可用于调节MSCs立体培养的良好生物相容性，更实现了一种有效抑制心肌梗死后心肌重塑的新方法。第二方面，我们尝试通过免疫手段改善心肌梗死局部炎症性单核细胞免疫学特点的方式来改善局部炎症情况，进而控制心肌重塑。我们将Ly6Chigh/low单核细胞与MSCs共培养72h；随后实验中我们在CX3CR1基因敲除背景小鼠基础上建立AMI模型后，给予或者不予MSCs移植治疗，发现 CX3CR1基因敲除小鼠AMI后循环中及梗死心肌局部Ly6Clow单核细胞数量很少，但是予MSCs移植后3天，循环中表达的Ly6Clow单核细胞的以及其核受体Nr4a1较前明显升高，而CX3CR1基因敲除小鼠AMI后3天，未行MSCs移植组，循环中表达的Ly6Clow单核细胞及核受体Nr4a1无明显变化，该发现直接说明Ly6Chigh单核细胞的表型转化可经免疫学手段进一步干预，进而改善心肌梗死后的心肌重塑，且该过程中的相关机制与核受体Nr4a1密切相关。进一步深入分析MSCS的本身特征发现其促进单核细胞表型转化与其分泌的外泌体以及外泌体来源的miR-150-5p密切相关。本课题关于心肌梗死后炎症单核细胞的免疫调节、纳米材料在AMI中的应用、改善MSCs疗效等工作的相关取得了重要成绩。

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