Gremlin1调控hiPSCs分化为内皮祖细胞在修复血管损伤中的作用及其机制

Endothelial progenitor cells (EPCs) have been shown to contribute to angiogenesis and tissue repair after ischemia. However, the number of endogenous EPCs is limited and the function is decreased in the disease state. Human induced pluripotent stem cells (hiPSCs) provide a good source of EPCs, and can achieve individualized treatment. At present, the mechanism of hiPSCs induced EPCs is not clear, and the efficiency is still to be improved. Our preliminary work confirmed that Gremlin1 could promote the repair of vascular injury caused by lower limb ischemia, which partly contributed to the recruitment of EPCs (Xiang QL, 2017) .Pre-experimental results showed that expression of Gremlin1 was obviously up-regulated during the differentiation of EPCs induced by hiPSCs. Knockdown of Gremlin1 by CRISPR/Cas9 technology significantly inhibited the differentiation. We will set up cell model of Gremlin1 knockout or overexpression and we plan to reveal the promoting effect and mechanism of Gremlin1 on EPCs formation, whicn through the antagonistic action of BMP and the activation of the VEGFR2 pathway. We also try to clarify the key role and significance of Gremlin1 in EPCs repair in the blood vessels. Finally, we will provide scientific basis for the treatment of ischemic vascular disease by hiPSCs/EPCs.

研究显示内皮祖细胞（EPCs）有助于缺血后的血管新生和组织修复。然而内源性EPCs数量受限，在疾病状态下功能下降。人诱导多能干细胞（hiPSCs）为EPCs提供了很好的来源。目前hiPSCs诱导成EPCs的机制尚未明确，效率仍有待提高。申请者前期工作证实，Gremlin1可促进下肢缺血的修复，该作用与EPCs募集有关（Xiang QL, 2017）；预实验结果显示，在hiPSCs诱导成EPCs的过程中，Gremlin1表达上调，运用CRISPR/Cas9技术敲除Gremlin1，分化显著受抑制。本研究将在细胞水平敲除或过表达Gremlin1，揭示Gremlin1通过拮抗BMP活性和激活VEGFR2通路，从而促进EPCs生成的作用及其机制；并在小鼠下肢缺血模型中，明确Gremlin1在EPCs修复血管中的关键作用和意义，最终为hiPSCs/EPCs治疗缺血性血管性疾病提供科学依据。

内皮祖细胞（EPCs）移植促进缺血部位的血管新生和组织修复，可用于缺血性疾病的治疗。然而内源性EPCs数量有限，在疾病状态下功能下降，无法满足治疗需求。人诱导多能干细胞（hiPSCs）为EPCs提供充足的来源，但hiPSCs诱导成EPCs的机制尚未明确，诱导效率有待提高。本研究首先从尿液中收集脱落的肾上皮细胞，建立人尿源性诱导多能干细胞（hUiPSCs）并诱导成为CD34+CD31+EPCs，作为缺血性疾病治疗的非侵入性内皮祖细胞来源。继而在细胞水平敲低或加入Gremlin1重组蛋白，发现Gremlin1在hUiPSCs向CD34+CD31+EPCs诱导分化过程的中胚层诱导阶段降低分化效率，而在CD34+CD31+EPCs诱导阶段和扩增阶段提高分化效率的阶段特异性作用。进而证明在扩增阶段添加外源性Gremlin1重组蛋白可促进CD34+CD31+EPCs的增殖，揭示了Gremlin1通过拮抗BMP活性和激活VEGFR2通路，从而促进CD34+CD31+EPCs生成的机制。动物实验方面，建立小鼠下肢缺血模型观察过表达Gremlin1蛋白的干细胞移植治疗下肢缺血的作用。结果显示，注射到缺血部位的细胞可以持续分泌高浓度的促血管生成因子Gremlin1，并通过激活经典的PI3K-AKT等信号通路促进血管生成、减轻缺血肌肉纤维化程度、促进组织损伤修复，从而提高肢体缺血性疾病治疗效果。本研究为EPCs提供了一个新的非侵入性来源，证明了Gremlin1在hUiPSC向CD34+CD31+EPCs分化过程中的关键作用，为改善缺血性疾病的干细胞疗法提供理论基础和技术支持。