药物联合MSCs移植趋化CXCR4+骨髓干细胞群持久靶向归巢修复梗死心肌及其机制研究

Based on our previous finding of Statins improving the effect of MSCs transplantation, the SDF-1 paracrine effect of MSCs/myocardial cells and the CXCR4+ stem cells' chemotaxis to SDF-1, we hypothesize that "Durgs combination with MSCs transplantation could persistently promote chemotaxis of bone marrow CXCR4+ stem cells' targeted homing to repair the infarcted myocardium". SDF-1 expression activator Resveratrol (RSV) and metablism inhabitor Sitagliptin are added to test their influence on MSCs in hypoxia/ischemia condition in vitro.Swine AMI model were created and randomized to Atorvastation (Ator), Ator+RSV, Ator+Sitagliptin, Ator+ RSV+Sitagliptin groups, combined with bone MSCs transplantation. The SDF-1 level, the quantity of implanted MSCs and targeted homing CXCR4+ stem cells, the myocardial perfusion, metablism and heart function will be evaluated and compared among groups in 1 and 6 weeks post infarction.Certify the above hyposis and clarify the mechanism of SDF-1 induced stem cells chemotaxis, has both scientific significance and application values for further study of stem cells and facilitating its therapeutic effect.

本研究基于前期"他汀提高骨髓间充质干细胞（MSCs）移植疗效"的突破性发现,以及MSCs/心肌细胞可旁分泌SDF-1且CXCR4+干细胞群具有强SDF-1趋化性的特点，提出"药物联合MSCs移植可趋化骨髓CXCR4+干细胞群持久性地靶向归巢修复梗死心肌"的假说。拟体外观察SDF-1表达增强剂白藜芦醇（RSV）及代谢抑制剂西格列汀对MSCs在缺血缺氧下分泌SDF-1的影响；建立猪急性心肌梗死（AMI）模型，随机接受骨髓MSCs移植联合：阿托伐他汀(Ator)、Ator+RSV、Ator+西格列汀或Ator+两药合用。在梗死后1周及6周检测SDF-1水平、心肌内移植MSCs数量及靶向归巢的CXCR+干细胞数量、心肌灌注/代谢及心功能变化，可望验证上述假说，并初步阐明药物联合MSCs移植对SDF-1诱导干细胞趋化作用的影响及其机制，对干细胞研究进一步深入及移植疗效的提高有重大科学意义及临床价值。

目的:明确阿托伐他汀(ATV) 预处理能否通过基质细胞衍生因子1(SDF-1)/ 趋化因子受体4(CXCR4)轴提高间充质干细胞(MSCs)的归巢能力，从而改善心梗后心功能。 方法：体外将SD大鼠MSCs 分为正常对照、ATV不同浓度与时间梯度组。流式细胞术和RT-PCR检测各组CXCR4表达，Transwell小室评估MSCs迁移能力。108只SD大鼠分假手术、AMI对照、MSCs移植与ATV预处理MSCs移植组(ATV-MSCs)。结扎前降支制作AMI模型，梗死后24小时尾静脉注射MSCs或ATV-MSCs (2×l06细胞/只)，对照组注射PBS。移植后3天及30天分别行心脏超声、病理组织学及分子生物学检测。结果：与正常对照相比ATV可以剂量依赖性的增加CXCR4表达，以1μM ATV处理组最显著(14.76 ± 3.05% vs. 1.98 ± 0.40%, P<0.001)；且呈一定的时间依赖性，其中处理12小时组CXCR4的表达至峰值(22.77 ± 2.03% vs. 2.20 ± 0.18%, P<0.001)，24小时仍保持在较高水平 (20.34 ± 4.13% vs. 2.20 ± 0.18%, P<0.001)。Transwell迁移结果显示，ATV预处理组MSCs向SDF-1迁移的数量是未处理组的2倍左右(24.65 ± 5.57 vs. 12.70 ± 2.40, P<0.001)，而加入CXCR4中和抗体后MSCs的迁移能力可被抑制。体内试验发现，细胞移植后3天 ATV-MSCs组归巢到梗死心肌区域的细胞数明显高于MSCs组(41.68 ± 10.80 vs. 65.30 ± 13.37, P<0.05)，在非梗死区域未发现移植的干细胞；移植后30天，ATV-MSCs组和MSCs组的存活率均低，MSCs组的存活率更低。与MSCs组相比，ATV-MSCs组的左室舒张末期内径和左室收缩末期内径减小，左室射血分数(62.28 ± 3.27% vs. 52.77 ± 7.05%，P=0.014)和左室短轴缩短率 (27.80 ± 2.16% vs. 22.27 ± 3.84%，P=0.015) 显著改善。结论：ATV预处理可通过提高MSCs表面CXCR4的表达而增强MSCs的迁移和归巢能力，是其改善心肌梗死后的心脏功能的机制之一。