CITED2在心脏干细胞衰老中的作用

The incidence and prevalence of cardiovascular diseases increase steeply with advancing age. Age-associated heart pathologies are thought to arise in part from degenerative changes in the organ's cellular compartment, primarily cardiac stem cells (CSCs). Currently, the molecular basis governing CSC aging remains unclear. Previous studies have demonstrated that CITED2, a transcription co-factor, is required for heart development and proper functioning. Mice deficient in CITED2 die at late gestation with cardiac malformation and other developmental defects. CITED2 mutations are found in patients with congenital heart disease. Our preliminary data indicate that CITED2 is strongly downregulated in aged CSCs, and its knockdown by shRNA cause cell growth arrest in vitro. Based on these findings, we propose to investigate the role of CITED2 in regulating CSC aging. To this end, the present project will focus on 1) addressing the age-dependent changes in the self-renewal capacity and differentiation potentials of mouse CSCs; 2) by utilizing gene modification techniques, determining the function of CITED2 in CSC maintenance with age and identifying mechanisms by which CITED2 modulates CSCs; 3) testing the ability of CITED2 to promote CSC-based repair of heart damage in vivo using a myocardial infarction animal model. The results from this application will not only have immediate implications for advancing our understanding of cardiac stem cell behavior change over age and underlying molecular mechanisms, as well as their potential involvement as prime instigators of heart failure and poor healing seen in the elderly, but also have tremendous potential in helping develop CSC replacement therapies or strategies to better maintain the pool of CSCs in situ for age-related heart diseases.

心脏干细胞（Cardiac stem cells，CSC）功能衰退是老年人群心脏病的主要内在原因之一。目前对CSC衰老的分子基础知之甚少。研究证明，CITED2是心脏发育和功能表现所必需的转录因子。CITED2敲除小鼠因心脏等器官发育缺陷而不能存活；CITED2基因突变则导致先天性心脏病。初步试验显示，CITED2在老龄小鼠CSC中的表达水平显著下降，实验性下调CSC的CITED2水平抑致细胞生长。基于这些证据，本项目拟探讨CITED2在心脏干细胞衰老中的作用；将开展三方面的工作：1）弄清CSC自我更新和分化潜能的年龄相关性变化规律；2）通过基因修饰技术确定CITED2对CSC功能的调节与机制；3）利用心肌梗死动物模型测定过表达CITED2对改善CSC心肌修复能力的效应。研究结果将提升对CSC衰老的行为变化及其调节机理的认识，并为发展年龄相关心脏疾病的CSC移植或在体介入处置策略奠定基础。

心脏干细胞（Cardiac stem cells，CSCs）与心脏功能维持及心脏疾病发生密切相关。本项目着重研究成体CSC衰老调节的分子基础。我们首先系统表征了小鼠CSCs。过去认为，小鼠CSCs为表达Sca-1的细胞。我们发现，小鼠心脏Sca-1+细胞可区分为Sca-1+CD31+和Sca-1+CD31-两个亚群。体外诱导实验表明，仅Sca-1+CD31-细胞能够分化形成心肌细胞、平滑肌细胞和血管内皮细胞。Macroarray分析显示， Sca-1+CD31-细胞呈现特征性的microRNA表达谱和转录组谱，其与Sca-1+CD31+细胞间存在170个miRNAs和1818个基因的表达差异（≥2倍）。这些差异表达的miRNAs和基因主要与增殖、分化和凋亡等相关，进一步确定了Sca-1+CD31-细胞的干细胞属性。随衰老小鼠CSCs数量上略有增加，但其自新和分化能力却下降，表明CSC功能随年龄增加而递减。CITED2在心脏早期发育中起重要作用，可能参与CSC衰老的调节；这是我们提出本项目的主要初衷之一。与青年小鼠相比（2-3月龄），老年小鼠（22-24月龄）Sca-1+CD31-细胞（或总Sca-1+细胞）中CITED2的表达水平下降50%左右，与CSC功能的衰退趋势一致。然而，敲低青年小鼠或过表达老年小鼠Sca-1+CD31-细胞的CITED 2并不影响这些细胞的增殖和存活。虽然CITED 2敲低或过表达可在一定程度上分别抑制和促进Sca-1+CD31-细胞的分化，但这些效应并不显著，说明CITED 2并非CSC衰老的关键调控因子。因此，我们转而比较分析了青年和老年小鼠Sca-1+CD31-细胞的转录表达谱。令人感兴趣的是，一些节律相关基因如Nr1d1 、Per2和DNA损伤诱导基因如Ddit4在两者间呈显著表达差异。这些基因可能作为重要组份构成CSC衰老的分子调控网络。概言之，本项目确定了小鼠CSCs存在于Sca-1+CD31-细胞中并筛选出了一些其功能调节的microRNAs和基因；阐释了小鼠CSCs年龄相关变化的规律与特征；明晰了CITED2在心脏发育和成体心脏功能维持中的不同地位或作用，初步鉴定出了一些其它的CSC衰老调控基因。这些结果或发现不仅有助于增进对心脏干细胞功能与衰老调节机制的了解，也将为心脏疾病的细胞病理基础及发展临床解决策略提供启发和基础。