基于微流控芯片平台模拟心肌梗塞微环境分选“优质”干细胞生物学特性和心肌梗塞疗效研究

Myocardial infarction is an important cause of morbidity and mortality among adults. Stem cell therapy is a promising modality for the treatment of myocardial infarction. However, most of the transplanted cells die within the first hours after transplantation because of hypoxia and oxidative stress; and the majority of cells after local intra-myocardial delivery migrate into non-targeted organs such as the lung, liver, and spleen. Therefore, the transplantation of stem cells into infracted hearts yields only marginal improvement in cardiac function.. Microfluidic device better mimicks the in vivo cellular microenvironment, generates stable chemoattractant gradients, and achieves high-throughput cell migration and isolation. Stromal cell-derived factor-1α (SDF-1α) and its cellular receptor CXCR4、vascular endothelial growth factor (VEGF) and its receptor VEGFR、tumor necrosis factor-α (TNF-α) and its cellular receptor TNFR-2 are known as the most prominent stem cell chemotaxis. SDF-1α、VEGF、TNF-α have been shown to be significantly up-regulated in myocardial infarction and attract the CXCR4+、VEGFR+、TNFR-2+ stem cells towards SDF-1α、VEGF、TNF-α gradients.. The present study utilizes microfluidic devices to create stable concentration gradient of SDF-1α、VEGF、TNF-α and myocardial infarction tissue extraction for selecting chemotactically and therapeutically potent stem cells for the treatment of myocardial infarction. Preconditioning with SDF-1α、VEGF、TNF-α suppresses stem cells apoptosis, enhances their survival, promotes their proliferation, enlarges their paracrine function, and improves their induction differentiation. Presumably, stem cells isolated on the basis of their chemotactic migration towards SDF-1α、VEGF、TNF-α demonstrate the significantly higher level of CXCR4、VEGFR and TNFR-2 receptor, and are more prone to survive、proliferate and secrete multiple cytokines. Over-expression of CXCR4、VEGFR and TNFR-2 enhanced migration、adhesion and engraftment of stem cells into infracted hearts. The number of surviving stem cells is positively proportional to their therapeutic effect. The present study investigates whether the chemotactically selected stem cells have the great abilities to improve cardiac function after transplantation into myocardial infarction.

心肌梗塞缺血缺氧及炎症细胞浸润导致移植干细胞凋亡死亡。心肌移植干细胞随血液淋巴循环迁移至外周器官。较低移植干细胞存活率和定植率是限制其心肌梗塞疗效的主要瓶颈。心肌梗塞上调SDF-1α、VEGF、TNF-α表达。SDF-1α/CXCR4、VEGF/VEGFR、TNF-α/TNFR-2轴调控干细胞趋化、迁移、定植于梗塞心肌。前期研究发现SDF-1α和梗塞心肌组织提取物高效趋化干细胞迁移，证实微流控芯片是高效干细胞分离筛选平台。据此提出应用微流控芯片技术，构建趋化因子配体（SDF-1α、VEGF、TNF-α）和梗塞心肌提取物浓度梯度，模拟心肌梗塞微环境，高通量分选趋化因子受体（CXCR4、VEGFR、 TNFR-2）阳性“优质”干细胞。趋化因子配体-受体结合诱导分选“优质”干细胞移植后迁移归巢于梗塞心肌，提高移植干细胞心肌定植率和存活率，促进干细胞修复心脏功能，探索提高干细胞心肌梗塞疗效新策略。

干细胞移植促进梗死缺血及周边区域毛细血管再生，增加左室射血分数。然而，心梗局部缺血缺氧，炎症微环境致移植干细胞凋亡死亡。干细胞移植后，通过淋巴和血液系统，引流至心脏外非靶向器官，存活率和靶向定植率较低，限制其疗效。.CXCR4/SDF-1α、Integrin β2/ICAM-1和Integrin α4/VCAM-1信号轴在干细胞趋化、粘附、迁移、归巢定植梗塞心肌过程中起关键调控作用。缺血缺氧和心肌坏死诱导细胞外基质和血管内皮SDF-1α、ICAM-1和VCAM-1表达增加。干细胞CXCR4或Integrin阳性率为～2%或5.11%，限制其疗效。基因转染和荧光细胞分选，构建CXCR4+或Integrin+细胞亚群。CXCR4配体与内皮细胞SDF-1α受体结合，Integrin配体与内皮细胞粘附分子受体结合，干细胞内皮粘附，透内皮层迁移，细胞外基质粘附，归巢定植。整合素是细胞膜表面跨膜蛋白，α和β亚基组成异二聚体，细胞内外信号传递媒介，调控细胞存活、增殖、迁移、分化和抗凋亡等特性。.脂肪干细胞（ASCs）是成体间充质干细胞，来源丰富，容易获取，扩增速度快。本课题调查: （1）梗塞心肌SDF-1α、ICAM-1和VCAM-1表达情况。（2） CXCR4、Integrin β2、Integrin α4-positive ASCs存活、增殖、迁移、分化、细胞因子分泌和抗凋亡特性。（3）CXCR4、Integrin β2、Integrin α4-positive ASCs 移植后，配体-受体介导，能否高效迁移、归巢定植于梗塞心肌？促进毛细血管再生，显著改善心脏功能？ .研究发现：（1） 心梗3-7d，SDF-1α、ICAM-1和VCAM-1表达水平升高。（2）CXCR4-positive ASCs心肌存活定植，小动物心脏超声CXCR4-ASCs移植组心功能显著优于ASCs对照组。（3）Integrin β2/integrin α4-positive ASCs增殖，存活，迁移能力强，诱导分化显著，分泌大量血管生长因子, 抗凋亡能力强。（4）13N-NH3·H2O PET/CT发现Integrin β2-positive ASCs改善梗塞心肌血液灌注。（5）18F-FDG PET/CT证实Integrin α4-positive ASCs显著减少心梗面积。