S1P联合PR-MSCs移植在治疗小鼠急性心肌梗死中的作用

Acute myocardial infarction (AMI), a main presentation of ischemic heart disease, is one leading cause of death globally. Although pharmacologic intervention and coronary artery bypass grafting, which can restore the blood flow (reperfusion), keep viable myocardium working, these approaches cannot rescue dying myocardium or restore normal cardiac functions. To use stem cell technology for the treatment of AMI has been one of the hot areas in cardiovascular research. Mesenchymal stem cells (MSCs) are primary seeding cells of heart transplantation used for the treatment of AMI. Accumulated evidence indicates that the transplantation of MSCs can effectively prevent ventricular remodeling, improve cardiac performance, and ameliorate the outcome of ST-elevated AMI. Our previous study found that, in comparison to the transplantation of the 5th MSCs, the transplantation of primary bone marrow mesenchymal stem cells (PR-MSCs) were more capable of rapidly migrating, proliferating in situ, and differentiating into mature vascular endothelial cells and cardiomyocytes. Meanwhile, PR-MSCs may contribute to the infarct-healing process and to improving post-MI cardiac performance through its paracrine effects on promoting cell migration, homing, and proliferation and neovasculature formation, and preventing apoptosis in the MI areas and border zones. However, the validity in repairing the injuried cardiomyocytes is limited bacause the numbers of live MSCs in situ are very small. Sphingosine 1 phosphate (S1P) can promote cell growth, inhibit apoptosis and promote angiogenesis through a variety of mechanisms. In this study, we will use S1P to pretreat PR-MSCs and hope that S1P can promote PR-MSCs proliferation, inhibit cell apoptosis, and maintain the stem cell properties. Furthermore, we can not only get a sufficient number of PR-MSCs in vitro, but also obtain more survived seeding cells in injured myocardium in situ after transplantation. So that the transplanted PR-MSCs can repair the injured myocardium and improve cardiac function more effectively in mice AMI models.

骨髓间充质干细胞能够通过向心肌细胞和血管内皮细胞分化、与原位心肌细胞融合、旁分泌作用以及激活内源性心脏修复机制来修复损伤心肌和改善心脏功能。我们前期研究证实：原代骨髓间充质干细胞(primary mesenchymal stem cells, PR-MSCs)凭借其优越的干细胞特性，在小鼠急性心肌梗死后具有更好的心肌修复作用及对于心功能有更强的改善能力。1-磷酸鞘氨醇(sphingosine 1 phosphate , S1P)能够通过各种作用机制发挥促进细胞生长、抑制细胞凋亡和促进血管新生的作用。我们利用S1P预处理PR-MSCs，期望S1P能够发挥促进PR-MSCs增殖，抑制其凋亡，并维持PR-MSCs的干细胞特性，不仅在体外能够得到数量足够的PR-MSCs，而且在移植后有更多的种子细胞在损伤心肌原位存活下来，以使得移植的PR-MSCs更好的发挥修复损伤心肌，改善心脏功能的作用。

小鼠原代骨髓间充质干细胞(primary mesenchymal stem cells, PR-MSCs)，凭借着优良的干细胞特性，与反复传代的骨髓间充质干细胞（bone marrow mesenchymal stem cells,BM-MSCs）相比，能在小鼠急性心肌梗死（AMI）后，更好地发挥修复损伤心肌及改善心脏功能的作用。但目前仍存在一个问题，即如何高效率地促进小鼠PR-MSCs的增殖与分化，以便获得更多干细胞移植的种子细胞。我们尝试运用一种具有多功能的磷脂分子——1-磷酸鞘氨醇（sphingosine 1 phosphate ,S1P），对小鼠PR-MSCs进行预处理，并评估S1P对小鼠PR-MSCs增殖、凋亡以及分化的影响。【研究内容】运用反复全骨髓转移的方法，在动物体外获取并扩增BM-MSCs；在PR-MSCs贴壁后，运用流式细胞仪，测定干细胞表面特异性标记分子；加入S1P预处理PR-MSCs，用IC50法测定实验浓度下S1P对PR-MSCs的细胞毒性；将 PR-MSCs 组细胞和 S1P-PR-MSCs组细胞置于培养箱中培养，用流式细胞仪对PR-MSCs的增殖进行分析；两组细胞贴壁后，继续培养7天，分别提取胞浆蛋白，采用 Western blot法，对VEGF,HGF,SDF,IGF-1,Caspase-3,bcl-2,bax等蛋白及Akt，ERK的磷酸化水平进行检测。【结果】S1P预处理后的PR-MSCs在缺氧24小时环境中的凋亡被抑制；S1P促进PR-MSCs经血管内皮生长因子（VEGF）诱导分化成血管内皮细胞，CD31与VCAM-1表达量在处理组中增加，表达VEGF、Angiopointin-1的基因在处理组中明显上调；S1P-PR-MSCs组中HGF, SDF-1, IGF-1等蛋白表达显著增加；S1P-PR-MSCs组中高表达P-ERK1/2，低表达caspase-3和Bax等细胞凋亡基因，而高表达Bcl-2等抗凋亡基因。【结论】S1P能显著抑制PR-MSCs在缺氧环境下的凋亡，促进PR-MSCs的增殖及经VEGF诱导分化为血管内皮细胞，并且S1P通过ERK通路影响PR-MSCs的旁分泌。