人心血管前体细胞移植对猪心梗后室性心律失常的影响及机制研究

Therapeutic studies of myocardial infarction (MI) with cardiovascular progenitor cells (CVPCs) have shown a clinical application potential, however, the effect of these cells to the MI-induced ventricular arrhythmias (VA) remains debate. The human CVPCs (hCVPCs) could be differentiated from human pluripotent stem cells (hPSCs) including embryonic stem cells (hESCs), which provide unlimited resources for the transplantable cells. We recently established a highly efficient differentiation system of hPSCs into ~90% of hCVPC. We confirmed that the hCVPCs can differentiate into enriched cardiovascular cells in vitro. We further demonstrated that the hCVPCs significantly improve the myocardial functional performance and reduce the fibrous scar after transplanted into infarcted mice hearts. However, whether hCVPCs can effectively control the VA occurring after MI needs to be investigated in the MI model from large animals. Therefore, we established the MI model in porcine by using percutaneous coronary balloon occlusion to minimize the damage to the porcine and to mimetic the events occurring in human to avoid permanent blocking the coronary vessels; the way for intracoronary injection of transplanted cells, and monitoring technology for the arrhythmia. In the present project, we aim to determine the following questions by the transplantation of hCVPCs into the porcine hearts with MI: (i) the effect of hCVPCs on the VA due to MI; (ii) the effects of hCVPCs on the post-MI myocardial function, infarcted area, and myocardial remodeling; and (iii) the mechanisms of cardiac protections of hCVPC transplantation on the MI-induced VA, myocardial performance, infarcted area, and myocardial remodeling. The findings from this study will provide new knowledge to the therapeutic strategy of stem cell transplantation, the control of MI-induced VA, and the myocardial repair.

心血管前体细胞(CVPCs)移植治疗心肌梗死(MI)可改善心功能,但对室性心律失常(VA)的作用尚未明确。CVPCs可由包括人胚胎干细胞(hESCs)在内的人多能干细胞分化获得。我们已成功将hESCs高效分化为90%以上CVPCs(hCVPCs),为心肌修复提供足量有前景的干细胞,并证实其在体外有效分化为心血管细胞、移植后显著减少MI鼠心梗面积及改善心功能。我们推测hCVPCs对MI后VA控制有效,但需大动物实验证实。为此我们建立了经皮冠脉内球囊封堵猪MI模型、冠脉内干细胞注射、心律失常监测等研究体系。本项目在猪MI后移植hCVPCs,拟明确:1)对VA及其基质有何影响？2)对心功能、心梗面积、心室重构等有何影响？3)调控猪MI后VA和心功能的机制如何？研究发现将揭示hCVPCs对MI后VA的影响和机制、发展治疗MI后VA及促进心肌修复的方法,并为治疗MI的移植细胞类型选择提供实验数据。

心肌梗死(MI)后并发心衰及室性心律失常的治疗仍面临重大挑战。本项目围绕着移植干细胞对MI后心肌修复及室性心律失常的效果作相关探索。用心导管介入技术的微创法成功制作猪MI模型，并用核磁共振及病理检测MI面积，并实施了室性心律失常检查方法。本项目也成功探索用微创法制作兔MI模型，通过向冠脉内放入弹簧圈或注入明胶海绵颗粒栓塞剂，制作兔MI模型，成功率达近60%。这种微创方法制作的兔MI模型可避免开胸损伤，更接近临床MI的病理特点。本项目也实现对兔进行心内膜电生理检查及心律失常模型制作的微创技术，在无需X线的条件下可进行兔心内电生理检查及心律失常模型制作，简单、易行、廉价、实用，可作用于心脏电生理科研、药物安全性检查、医疗器械研发等，具有较大的科研价值。本项目也探索了经静脉注射间充质干细胞对兔MI后心肌修复，发现静脉注射5×106以上的间充质干细胞可以显著改善MI后的心肌修复。.在前期已经建立了能达到90%纯度的心血管前体细胞(CVPCs)分化体系基础上，我们进一步优化分化体系，将人多能干细胞衍生CVPCs分化效率提高到95%以上。构建了携带绿色荧光蛋白(GFP)报告基因的诱导人多能干细胞系(hiPSC-GFP)，构建的hiPSC-GFP能被高效诱导分化为CVPCs和心肌细胞。将hiPSC-GFP分化的CVPCs移植到小鼠MI模型中可显著改善小鼠MI后14天和28天心功能，减少心脏纤维化及疤痕形成；进一步研究发现移植的CVPCs可分化为心肌细胞。这些研究进一步明确了人多能干细胞衍生CVPCs的心肌修复作用，为进一步机制研究提供了新的线索。本项目也筛选获得调控胚胎干细胞向心肌细胞分化的潜在miRNA。揭示miR-142-3p在早期心肌细胞分化中的重要作用：miR-142-3p通过抑制心脏中胚层形成而抑制心肌细胞分化，心脏转录因子Mef2C是miR-142-3p的靶基因。该研究为理解miRNA对胚胎干细胞向心肌细胞分化的调控机制提供了新的线索，有助于更好地了解在体心脏发育和心肌细胞形成过程。