Periostin调控ADSC存活、迁移及促进损伤血管再内皮化的机制研究

Endothelial cell injury or dysfunction is one of the primary reason of restenosis and thrombosis after angioplasty of cardiovascular diseases. Therefore, how to promote the re-endothelialization of injured arteries as early as possible will be of great significance. Adipose derived stem cells (ADSC) played a vital role in promoting re-endothelialization of injured arteries, which shows a wide prospect of clinical application. However, our previous results showed that ADSC weakened in the target vessel parts of the survival, migration and differentiation capacity, which is the key to affecting our active effect. Periostin protein as a member of the ECM family has strong antiapoptotic, promoting migration and angiogenesis in tumor cells and vascular endothelial cells, but its expression is lower in ADSC. Thus we propose a hypothesis that overexpression of periostin by transfection on ADSC may be better to promote its survival, migration and improve re-endothelialization ability. In this study, nuclear transfection, RNA Interference Silencing and Receptor Blocking technologies will be adopted to make ADSC overexpressing or down-regulation periostin, and GFP gene was used for tracing the role of ADSC in promoting re-endothelialization in an Apo E model of carotid artery injury after transplantation. After further observing the influence of periostin on ADSC survival, migration and differentiation capacity, the associated integrin/TGF β/FAK/Akt signal pathway were discussed, finally elucidate the molecular mechanism of how periostin regulates ADSC survival, migration and promotes re-endothelialization, which will bring new insights into the treatment of restenosis and thrombosis after angioplasty.

内皮细胞损伤或功能障碍是腔内血管重建术后再狭窄和血栓形成的重要原因，尽早促进损伤血管再内皮化具有重要意义。脂肪干细胞（ADSC）移植促进再内皮化已显示较好应用前景，但我们前期研究表明ADSC在靶血管部位存活、迁移及分化能力减弱，是影响疗效的关键所在。Periostin作为ECM家族中的一员，在肿瘤和内皮细胞中有强大的抗凋亡、促迁移和血管新生的作用，但在ADSC中表达较低。由此我们提出假设：通过使ADSC过表达periostin，可能会更好的促进其存活、迁移及提高再内皮化能力。本课题拟采用核转染、RNA干扰、受体阻断等技术使ADSC过表达或沉默periostin，利用GFP基因示踪，移植修复ApoE小鼠颈动脉内皮损伤，观察periostin对ADSC存活、迁移和再内皮化的影响，探讨相关联的整合素/TGFβ/FAK/Akt信号通路，阐明其分子机制，为防治血管重建术后再狭窄和血栓形成提供新思路。

本研究旨在探索Periostin调控ADSC存活、迁移及促进损伤血管再内皮化的作用及其机制。我们从C57及GFP小鼠获取了ADSC细胞，并通过腺病毒过表达技术成功建立了高表达Periostin基因的ADSC体系Periostin-ADSC（P-ADSC）。.首先，我们验证了Periostin对ADSC存活、迁移、粘附、增殖及分化能力的影响。我们发现Periostin转染更够显著抑制ADSC在低氧条件下的凋亡。我们利用Transwell试验证实Periostin能够显著促进ADSC的迁移。粘附实验进一步证实转染Periostin可以明显的促进ADSC的粘附能力。此外，我们发现Periostin能够促进ADSC的多向分化能力。 并且，Periostin转染能够促进ADSC分泌VEGF、HGF、IGF-1及bFGF等生长因子的能力，说明Periostin可能通过促进ADSC的旁分泌作用来促进ADSC的再内皮化或血管化。.其次，我们对Periostin调控ADSC存活、迁移及促进损伤血管再内皮化的机制进行了深入研究。我们分析了正常氧环境和低氧环境下P-ADSC和ADSC中与凋亡相关蛋白Bcl-2和Bax的表达，结果显示：P-ADSC的抗凋亡蛋白Bcl-2显著增加而促凋亡蛋白Bax显著降低，说明Periostin具有抑制低氧环境下的细胞凋亡的作用。 我们进一步分析发现，P-ADSC中 integrin-β1/FAK/PI3K/Akt/eNOS 信号通路蛋白与ADSC相比可维持更长时间的磷酸化形式，且表达量显著上调。该研究结果表明periostin通过调节integrin-β1/FAK/PI3K/Akt/eNOS通路促进ADSC存活、黏附、迁移及分化。. 最后，我们成功建立了Apo E小鼠下肢缺血模型并进行干细胞移植。我们发现在干细胞移植4周后，P-ADSC组缺血下肢血流灌注指数显著优于ADSC和空白对照组。组织学分析显示P-ADSC和ADSC在缺血肢体中逐渐分化为内皮细胞，微血管密度定量分析显示P-ADSC组显著高于ADSC和空白对照组。α-actin免疫荧光染色，CD31免疫组化染色，以及HE染色均证实 P-ADSC 组血管化水平显著高于ADSC组。我们的研究结果表明Periostin能够调控ADSC存活、迁移及促进损伤血管再内皮化，因此有望用于下肢缺血性疾病的治疗。