CGRP调节NF-κB信号通道促进心肌梗死后c-kit+心脏干细胞存活及分化的研究

Myocardial infarction, one of the severe ischemic diseases, has few great progress in the therapy on improving cardiac function. Recently most studies have demonstrated that the adult mammalian heart contains a population of undifferentiated c-kitpos Linneg cells(c-kitpos CSCs), which are self-renewing, clonogenic ,and multipotent, giving rise to restore the infarted myocardium. However, the local microenvironment after myocardial infarction, including oxygen stress and inflammation, can lead to the transplanted or migrated cell undergoing apoptosis, making the utilization of transplanted or migration cell very low. NF-κB signaling pathway plays a key role in regulating the inflammatory microenvironment and apoptosis after myocardial infarction. Our previous study indicated CGRP can improve cardiac function after myocardial infarction by inhibiting NF-κB activation. Thus, we attempts to investigate whether CGRP can improve the survival and differentiation of CSCs after myocardial infarction by regulating NF-κB signaling pathway. In order to confirm this hypothesis, we study the effects of CGRP on CSCs survival and differentiation both on cell oxidative stress model in vitro and myocardial infarction model in vivo. Our experiment on correlations between NF-κB and CGRP is to reveal the mechanisms as well as accumulate experimental evidence and provide new methods for clinical stem cell therapeutics in myocardial infarction.

心肌梗死后的心脏修复治疗是临床亟待解决的重大问题。多项研究提示，成年哺乳动物心脏中含有未分化的c-kit+心脏干细胞（c-kit+CSCs），对其激活及利用有助于梗死后心脏修复。然而梗死局部炎症和氧化应激的微环境，导致细胞利用率极低。我们既往研究显示RAMP1可上调CGRP的效应性，抑制NF-κB信号通路，从而改善梗死区域的炎症微环境。结合预实验结果，我们推测:CGRP可能具有增强内源性CSCs抵御局部微环境能力并促进细胞存活及分化。为验证此假设，本项目建立体外细胞氧化应激和大鼠心肌梗死模型，从不同角度观察CGRP对心肌梗死后氧化应激状态下c-kit+CSCs的存活及分化能力的影响，并检测此过程中NF-κB 相关信号蛋白和凋亡相关基因的表达水平，研究CGRP对CSCs的存活及分化的调节作用及其与微环境中NF-κB信号通道的关系，以期为提高干细胞移植治疗效果提供新的思路。

心肌梗死后的心脏修复治疗是临床亟待解决的重大问题。多项研究现示，成年哺乳动物心脏中含有未分化的c-kit+心脏干细胞（c-kit+CSCs），对其激活及利用有助于梗死后心脏修复。然而梗死局部炎症和氧化应激的微环境，导致细胞利用率极低。我们既往研究显示受体活性修饰蛋白1（RAMP1）可上调降钙素基因相关肽（CGRP）的效应性，抑制核转录因子kappa B（NF-κB）信号通路，可改善梗死区域的炎症微环境。结合前期实验结果，我们推测:CGRP可能具有增强内源性c-kit+CSCs抵御局部微环境能力并促进细胞存活及分化作用。本研究结果显示:在缺氧状态下，NF-κB信号通路被激活，荧光显微镜明显观察到NF-κBP65发生核转位，NF-κB相关信号蛋白P-I-κB、NF-κBP65及NF-κBP50表达明显，应用CGRP干预后缺氧状态下c-kit+CSCs 细胞NF-κB相关信号蛋白表达减少；为了进一步验证CGRP调节缺氧状态下c-kit+CSCs的NF-κB信号通路，使用CGRP阻滞剂CGRP8-37抑制CGRP作用后，NF-κB相关信号蛋白表达增加；同时观察到CGRP干预后缺氧状态下c-kit+CSCs增殖存活能力增强，早、晚期细胞凋亡率均降低，这说明NF-κB信号通路对缺氧状态c-kit+CSCs增殖、存活及凋亡具有调剂作用。但实验中抑制缺氧状态下c-kit+CSCs的NF-κB信号通路后，只是在一定程度上降低凋亡率，这说明有其它信号通路参与细胞的增殖凋亡过程，本实验进一步研究发现，PTEN/PI3K/AKT信号通路在c-kit+CSCs增殖、凋亡中也起了重要作用，NF-κB信号通路和PTEN/PI3K/AKT信号通路相互交织参与氧化应激状态下细胞的增殖存活通路，CGRP对CSCs的增殖存活的调节作用及其与微环境中NF-κB信号通道和PTEN/PI3K/AKT信号通路的调节作用，这些研究结果为提高干细胞移植治疗心肌梗死的研究提供新的思路。