补气活血中药优化微环境在诱导多功能干细胞移植治疗心肌梗死中的作用机制

Transplantation of induced pluripotent stem cell (iPSC) could produce positive improvement on cardiac function after myocardial infarction (MI). However, the survival and differentiation of stem cells after transplantation was limited by the hypoxia-ischemia microenvironment. Our previous study found that Chinese medicine for replenishing qi and activating blood could optimize the microenvironment and improve the survival and differentiation of stem cells. However, the mechanisms on survival and differentiation of stem cell regulated by Chinese medicine are not very clear. This project intends to, on the basis of Chinese medicine promoting the effect of iPSC transplantation in the treatment of MI, select active ingredient enhancing the survival of iPSC and inducing the differentiation of iPSC into cardiomyocytes through specific signal luciferase reporter gene, cardiac cell-specific promoter TNNT2-luciferase reporter gene and TNNT2-GFP iPSC line, to investigate the effect of transplantation of iPSC pretreated with active ingredient from Chinese medicine, and to explore the mechanisms of PI3K/Akt and Wnt-11/JNK signaling pathway in Chinese medicine regulating survival and differentiation of iPSC through lentiviral gene overexpression and RNA interference technologies. This study could not only provide strategy and means to solve the problem of stem cell transplantation faces, but also provide theoretical basis and practical basis for the clinical application of stem cell transplantation regulated by Chinese medicine in the treatment of myocardial infarction.

诱导多功能干细胞(induced pluripotent stem cell, iPSC)移植可改善心肌梗死后心功能，但心肌梗死后局部缺血缺氧微环境不利于干细胞的存活和分化。我们研究发现补气活血中药可优化移植微环境，促进干细胞的存活和分化，然而中药调控干细胞存活和分化的作用机制尚不清楚。本课题拟在补气活血中药促进iPSC移植治疗心肌梗死的基础上，通过特异信号通路荧光素酶报告基因、启动子荧光素酶报告基因和启动子控制表达的荧光报告iPSC细胞系筛选促进 iPSC存活和诱导iPSC向心肌细胞分化的有效成分，研究中药有效成分预处理iPSC移植治疗心肌梗死的作用，并通过基因过表达和基因沉默技术探讨PI3K/Akt和Wnt-11/JNK信号通路在中药调控iPSC存活和分化中的作用机制，不仅为解决干细胞移植面临的问题提供策略和手段，也为临床应用中药调控干细胞移植治疗心肌梗死提供理论依据和实验基础。

诱导多能干细胞（induced pluripotent stem cell, iPSC）移植可改善心肌梗死后心功能，但心肌梗死后局部微环境不利于干细胞的存活和分化。我们研究发现补气活血中药可优化移植微环境，促进干细胞的存活和分化，然而中药调控干细胞分化和存活的作用机制尚不清楚。本课题拟在补气活血中药促进iPSC移植治疗心肌梗死的基础上，通过启动子荧光素酶报告基因筛选促进 iPSC存活和向心肌细胞分化的有效成分，进一步研究中药有效成分预处理iPSC移植治疗心肌梗死的作用，并通过基因过表达和基因沉默技术探讨Wnt-11/JNK信号通路在中药调控iPSC分化的作用机制。结果发现，加味桃红四物汤能够改善心肌梗死后心功能，并可通过优化微环境促进iPSC移植治疗心肌梗死的作用。杨酶酮、二氢丹参酮I、黄芪甲苷、0.1 uM 三七皂苷Fe、1 uM 三七皂苷R1（Notoginsenoside R1, NGR1）、白藜芦醇和淫羊藿苷等可以激活Myh6，但低浓度的三七素和高浓度的三七皂苷Fe可以抑制Myh6。淫羊藿苷和白藜芦醇诱导iPSC向心肌细胞分化的效率最高，两者联合诱导可促进iPS来源的心肌细胞（iPS derived cardiomyocytes, iPS-CMs）的成熟。通过PI3K 信号通路荧光素酶报告基因系统发现，NGR1可以激活PI3K/Akt信号通路，有利于干细胞的存活。在此基础上，进一步研究NGR1预处理的间充质干细胞（Mesenchymal stem cells, MSCs）和iPS-CMs移植治疗心肌梗死的作用及可能机制。NGR1预处理可促进MSCs移植后的存活，降低梗死面积和梗死区胶原纤维沉积，并促进梗死区血管新生和旁分泌。此外，NGR1预处理可以促进移植iPS-CMs的存活和旁分泌，增强梗死区血管密度和淋巴管密度，从而改善心功能和降低梗死面积。并发现Wnt11/JNK信号通路在中药有效成分诱导iPS-CMs分化和成熟中发挥一定的调控作用。本研究可为促进干细胞存活和向心肌细胞定向分化提供新的小分子物质，为丰富和阐明iPSC诱导分化的作用机制提供实验数据，并为中西医结合治疗心肌梗死等缺血性心肌病提供了新的策略和手段。