背景:羊膜来源的人羊膜间充质干细胞(hAMSCs)具有多谱系分化,免疫抑制,和抗炎使它们适合治疗各种疾病。
目的:本研究旨在探讨hAMSCs在心室重构(VR)中的作用及其分子机制。
方法:通过流式细胞仪分析等一系列实验对hAMSCs进行表征,免疫荧光,分化诱导和致瘤性。异丙肾上腺素(ISO)腹膜诱导小鼠VR模型,并通过超声心动图评价hAMSCs移植的治疗效果和潜在机制,羧基荧光素二乙酸酯琥珀酰亚胺酯(CFSE)标记的细胞示踪,组织化学,qRT-PCR和蛋白质印迹分析。进行了共培养实验,以进一步探索hAMSCs来源的条件培养基(CM)对巨噬细胞极化和成纤维细胞纤维化的作用机制。
结果:hAMSCs移植可明显减轻ISO诱导的VR,包括心肌肥厚和纤维化,改善心功能。CFSE标记的hAMSCs在心脏中保持未分化状态,表明hAMSCs介导的ISO诱导的VR改善可能与其旁分泌效应有关。hAMSCs显著抑制ISO诱导的炎症和纤维化,M2巨噬细胞浸润增加,CD206、IL-10表达增加,CD86、iNOS,COL3和αSMA在心脏中的表达,提示hAMSCs移植促进M2巨噬细胞的极化,抑制M1巨噬细胞的极化。机械上,在体外培养的RAW264.7巨噬细胞中,hAMSCs来源的CM显著增加CD206、IL-10、Arg-1的表达,降低iNOS和IL-6的表达。有趣的是,RAW264.7-CM显著促进IL-10、IDO、和COX2在hAMSCs中。此外,用RAW264.7-CM预处理的hAMSCs来源的CM显著抑制3T3细胞中αSMA和COL3等纤维发生基因的表达。
结论:我们的结果表明,hAMSCs有效减轻ISO诱导的心肌肥厚和纤维化,改善了小鼠的心脏功能,其潜在机制可能与通过促进心脏组织CD206hiIL-10hi巨噬细胞的极化抑制心室重构过程中的炎症和纤维化有关。我们的研究强烈表明,通过利用有效的免疫抑制和抗炎作用,hAMSCs可能为临床上预防和治疗VR提供替代治疗方法。
BACKGROUND: Human amniotic mesenchymal stem cells (hAMSCs) derived from amniotic membrane have multilineage differentiation, immunosuppressive, and anti-inflammation which makes them suitable for the treatment of various diseases.
OBJECTIVE: This study aimed to explore the therapeutic effect and molecular mechanism of hAMSCs in ventricular remodeling (VR).
METHODS: hAMSCs were characterized by a series of experiments such as flow cytometric analysis, immunofluorescence, differentiative induction and tumorigenicity. Mouse VR model was induced by isoproterenol (ISO) peritoneally, and the therapeutic effects and the potential mechanisms of hAMSCs transplantation were evaluated by echocardiography, carboxy fluorescein diacetate succinimidyl ester (CFSE) labeled cell tracing, histochemistry, qRT-PCR and western blot analysis. The co-culturing experiments were carried out for further exploring the mechanisms of hAMSCs-derived conditioned medium (CM) on macrophage polarization and fibroblast fibrosis in vitro.
RESULTS: hAMSCs transplantation significantly alleviated ISO-induced VR including cardiac hypertrophy and fibrosis with the improvements of cardiac functions. CFSE labeled hAMSCs kept an undifferentiated state in heart, indicating that hAMSCs-mediated the improvement of ISO-induced VR might be related to their paracrine effects. hAMSCs markedly inhibited ISO-induced inflammation and fibrosis, seen as the increase of M2 macrophage infiltration and the expressions of CD206 and IL-10, and the decreases of CD86, iNOS, COL3 and αSMA expressions in heart, suggesting that hAMSCs transplantation promoted the polarization of M2 macrophages and inhibited the polarization of M1 macrophages. Mechanically, hAMSCs-derived CM significantly increased the expressions of CD206, IL-10, Arg-1 and reduced the expressions of iNOS and IL-6 in RAW264.7 macrophages in vitro. Interestingly, RAW264.7-CM remarkably promoted the expressions of anti-inflammatory factors such as IL-10, IDO, and COX2 in hAMSCs. Furthermore, the CM derived from hAMSCs pretreated with RAW264.7-CM markedly inhibited the expressions of fibrogenesis genes such as αSMA and COL3 in 3T3 cells.
CONCLUSIONS: Our results demonstrated that hAMSCs effectively alleviated ISO-induced cardiac hypertrophy and fibrosis, and improved the cardiac functions in mice, and the underlying mechanisms might be related to inhibiting the inflammation and fibrosis during the ventricular remodeling through promoting the polarization of CD206hiIL-10hi macrophages in heart tissues. Our study strongly suggested that by taking the advantages of the potent immunosuppressive and anti-inflammatory effects, hAMSCs may provide an alternative therapeutic approach for prevention and treatment of VR clinically.