miR-210在心肌干细胞移植治疗心肌梗死中的调控作用及机制研究

Ischemic heart disease with a high mortality is facing the lack of effective treatment. Transplantation of cardiac stem cells (CSCs) has been emerging as a research focus. However, the most serious problem in this field is that the protective effects are severely restricted by poor survival, due to limited hypoxia tolerance of CSCs. In our previous studies, it is identified that hypoxia-preconditioning downregulated the expression of Casp8ap2 and upregulated expression of CXCR4 of CSCs, consequently enhanced their survival and migration. And the further research revealed that miR-210 was also upregulated in this procedure. More importantly, survived CSCs have a significantly higher level of expression of miR-210 than apoptotic ones have, after sorted by FACS with annexin V. Additionally, increased ability of anti-hypoxia and migration was detected in miR-210 overexpressed CSCs, which was reversed by anti- miR-210. It has been currently documented that miR-210 is an important hypoxia-regulated microRNAs, protecting cells against hypoxic injury. Therefore, it is speculated that miR-210 may improve CSCs ability of anti-hypoxia via regulating apoptosis-related genes. The aim of this study is to confirm miR-210 as a key factor of CSCs against hypoxia, and to research which downstream genes may be involved in survival and migration of CSCs. This study may provide a new therapeutic strategy for ischemic heart disease.

缺血性心脏病致死率高，且缺少有效治疗手段。心肌干细胞移植是治疗研究热点，但面临的最大问题是其无法耐受移植区缺氧环境，低存活率严重影响疗效。我们既往研究证实，低氧预处理心肌干细胞通过下调Casp8ap2、促进CXCR4表达增强其植入后生存及迁移能力，但具体机制不明。microRNA芯片结果提示低氧处理后miR-210上调明显，更有意义的是，缺氧条件下miR-210表达在存活心肌干细胞中显著增加（对比凋亡细胞）。将miR-210类似物转入可增强心肌干细胞抗凋亡及迁移能力，转入miR-210抑制子则相反。miR-210是新近发现的缺氧诱导miRNAs中重要一员，保护细胞抵抗缺氧。因此，miR-210可能在心肌干细胞抗缺氧中起重要作用。本研究旨在明确miR-210是心肌干细胞抵抗缺氧的关键因子，明确miR-210通过何种机制促进心肌干细胞存活及迁移，研究结果将为缺血性心脏病治疗带来新希望。

背景：心肌干细胞（Cardiac stem cells, CSCs）是治疗心肌梗死理想的种子细胞。本课题旨在揭示缺氧相关miR-210对CSCs的保护机制，并观察过表达miR-210的CSCs在急性心肌梗死中的治疗作用。方法：取SD大鼠 CSCs体外培养至第4代。用携带miR-210模拟物（CSCsmiR-210）和抑制物（CSCsanti-miR-210）的慢病毒转染CSCs，并用空病毒作对照。1周后，检测缺氧条件下Casp8ap2、Caspase 8、PTPN2、CXCR4、HIF-1α的表达，并测定CSCs迁移、凋亡以及心肌细胞分化和细胞因子分泌情况。将大鼠急性心梗模型随机分为4组（每组n=20）：对照组、CSCs组、CSCsanti-miR-210组、CSCsmiR-210组。在后三组中，于梗死区域直接心肌内注射5 × 106个CSCs。1天后，检测植入CSCs凋亡以及细胞因子表达。4周后，评价局部增殖、血管再生、移植CSCs心肌细胞分化、心肌密度、左室功能及重构。结果：体外发现，缺氧诱导CSCs中miR-210表达。与未处理CSCs相比，CSCsmiR-210中Casp8ap2、Caspase8、PTPN2和HIF-1α表达明显上调（P < 0.05），但CXCR4无变化（P > 0.05）；凋亡明显减低；迁移明显增加；另外，心肌特异性蛋白及细胞因子表达明显增高且能被HIF-1α抑制剂逆转（P < 0.05）。而在CSCsanti-miR-210中结果相反（P < 0.05）。在体发现，与对照组和CSCs组相比，CSCsmiR-210组中移植CSCs凋亡明显减少且向心肌细胞分化增强；局部IGF-1和VEGF表达明显增加，细胞增殖、血管再生、心肌密度均明显增多；左室射血分数和舒张末内径明显改善（P < 0.05）。CSCsanti-miR-210组结果相反（P < 0.05）。科学意义：本研究证实，miR-210是CSCs中一个关键的缺氧调控因子。过表达的miR-210不仅通过Casp8ap2/Caspase 8 and PTPN2抑制CSCs凋亡，促进其迁移；而且通过调控HIF-1α促进CSCs心肌细胞分化功能和旁分泌功能。过表达miR-210的移植后CSCs明显改善心功能，抑制心室重构，可能成为缺血性心肌病一种新的治疗策略。