Sirt1通过去乙酰化Sox2逆转脂肪干细胞衰老及改善哮喘治疗的研究

Adipose-derived stem cells (ASCs) have many advantages, such as being easy to acquire, steady curative effect and having no significant side effect，which have made them an ideal cell source for asthma therapy. However, ASCs undergo senescence with the in vitro culture，coupled with the decrease of self-renewal ability and differentiation potential, which become the bottleneck restricting curative effect in anti-asthmatic treatment. In previous study, we have observed that the deacetylase Sirt1 expression decreased in the senescent ASCs. We also demonstrated that Sirt1 interacted with Sox2 through Oct4 in embryonic stem cells. Considering the central role of Sox2 in maintaining ASCs self-renewal and pluripotency, we hypothesis that up-regulating Sirt1 will enhance Sox2 transcriptional activity mediated by Oct4 to improve self-renewal and pluripotency in senescent ASCs, and improve therapeutic potential of ASCs for anti-asthmatic treatment. In this project, we will demonstrate that Sirt1 form complex with Oct4-Sox2 and regulate the transcriptional activity of Sox2 in ASCs; Furthermore, we will demonstrate the influence of Sox2 transcriptional activity variation on the role of Sirt1 maintaining self-renewal and differentiation potential in senescent ASCs; In addition, we will transplant Sirt1-engineered senescent ASCs to asthma model mice, and estimate the curative effect as well as tracking ASCs fates. This study is expected to provide a potential target and experimental basis for improving the therapeutic potential of ASCs in anti-asthmatic treatment.

脂肪干细胞（ASCs）治疗哮喘具有取材方便，疗效稳定无副作用的优点，但ASCs易于随着体外扩增衰老，自我更新能力和分化潜能下降，成为制约哮喘疗效的瓶颈。我们前期在衰老的ASCs发现去乙酰化酶Sirt1表达下降及在胚胎干细胞发现Sirt1可通过Oct4与Sox2相互作用。基于Sox2在ASCs维持自我更新和分化潜能的核心作用，我们假设：在衰老ASCs中提高Sirt1的表达，可通过Oct4介导Sox2去乙酰化，激活Sox2转录活性，恢复细胞自我更新和分化潜能，改善哮喘疗效。本研究拟在分子水平证明ASCs中Sirt1与Oct4-Sox2形成复合物，并调节Sox2的转录活性；在细胞水平明确Sox2转录活性对Sirt1改善衰老ASCs自我更新和分化潜能的影响；用sirt1基因修饰的ASCs移植给哮喘小鼠，观察治疗作用，同时追踪ASCs命运。本项目将为改善哮喘的ASCs治疗提供新的分子靶点和实验依据。

项目背景：脂肪间充质干细胞（ASC）治疗哮喘有显著的疗效。但是目前自体ASC治疗面临细胞衰老的瓶颈问题：每次分离的ASC数量很少，要达到千万级的治疗用量，需要经过长时间体外扩增。体外扩增导致细胞衰老和细胞多潜能性下降。本研究的主要目的是通过Sirt1基因修饰防止ASC衰老，并研究Sirt1抗ASCs衰老的分子机制。. 本项目的主要研究内容包括：在分子水平研究了Sirt1与Sox2的相互作用；在细胞水平研究Sirt1对ASCs细胞增殖和多潜能性的影响；在动物水平，通过制备小鼠支气管哮喘模型，观察静脉注射Sirt1基因修饰ASCs是否能够提高哮喘的疗效。. 通过以上研究，本研究主要获得的结果有：研究了Sirt1与Sox2的相互作用，并发现这种相互作用是依赖Oct4完成的；分离鉴定了小鼠ASCs并观察了ASCs的衰老过程，发现小鼠ASCs一般第7代出现增殖缓慢，第9代出现明显衰老特征，第14代β-半乳糖苷酶染色阳性明显，第17代β-半乳糖苷酶染色阳性细胞达到80%，第19代停止增殖并逐渐死亡。我们制备了Sirt1上调与下调的ASCs，发现Sirt1上调的细胞明显逆转衰老，其细胞形态与增殖能力均优于野生对照组；我们制备了小鼠支气管哮喘模型，通过注射Sirt1基因修饰或野生型ASCs，观察哮喘症状和肺组织炎症评分，发现注射Sirt1基因修饰ASCs组小鼠喘息症状较轻微，肺组织切片表现出较低的炎症评分，PAS染色显示出更好的粘液分泌控制作用。. 我们的结果提示：Sirt1是抗ASCs衰老的重要分子，上调Sirt1的表达水平可提高ASCs对哮喘的抗炎疗效。本研究基本按照计划执行，完成了既定的目标，累计发表论文22篇，其中SCI收录论文19篇，中文期刊3篇。课题组成员共参加国内外学术会议7人次。投入直接研究经费55万元，支出54.9887万元，剩余113元。.