基于衰老的细胞模型研究miR-34a-Nampt调控网络在大鼠间充质干细胞衰老中的作用

Organism aging has been attributed to the stem cells senescence. This is an essential scientific issue in the field of gerontology and stem cell research. As a rate-limiting enzyme in NAD+ biosynthesis, nicotinamide phosphoribosyltransferase (Nampt) determines NAD+ levels and further regulates Sirt1 activity. The network is named as Nampt-NAD-Sirt1 axis, which plays an important role in cellular metabolism, cell differentiation and aging. Our previous study found that the senescence of rat MSCs occurred during the serial cultivation in vitro. Nampt expression in MSCs from aged individuals was reduced, while miR-34a expression was upregulated. The bioinformatics analysis demonstrated that miR-34a had a binding site with the 3'-UTR region of Nampt. However, whether MSCs senescence is able to be influenced by Nampt and whether its role can be regulated by miR-34a remain unclear. This project therefore intends to investigate the effect of Nampt on MSCs senescence and its regulatory mechanism by miR-34a-Nampt network by the use of MSCs replicative senescence model in vitro and MSCs normal aging model in vivo. It will provide important theoretic and experimental basis for delaying stem cells senescence and prevention and treatment of age-related diseases.

生物体的衰老是由于自身的干细胞衰老而引起的，这是老年医学和干细胞研究领域中重大的科学问题。Nampt是NAD+生物合成的限速酶，决定代谢中NAD+水平和Sirt1活性，称为Nampt-NAD-Sirt1轴，对细胞代谢、细胞分化及生物体衰老起重要作用。我们发现：体外连续传代培养使大鼠MSCs出现衰老；衰老个体MSCs中Nampt表达降低，而miR-34a表达上调；生物信息学网站预测miR-34a存在与Nampt 3'-UTR的结合区域。然而Nampt对MSCs衰老是否有作用，其作用是否受miR-34a调控均不清楚。本项目拟通过体外大鼠MSCs复制性衰老模型和体内MSCs自然衰老模型，利用基因转染、RNAi技术及双荧光素酶报告基因实验等方法，研究Nampt对MSCs衰老的作用，进一步探讨miR-34a-Nampt网络在干细胞衰老中的调控机制，为延缓干细胞衰老及防治各种老龄化疾病提供重要依据。

Nampt是NAD+生物合成的限速酶，决定代谢中NAD+水平和Sirt1活性，称为Nampt-NAD+-Sirt1轴，对细胞代谢、细胞分化及生物体衰老起重要作用。然而Nampt对大鼠MSCs（rMSCs）细胞衰老是否有作用，其作用是否受miR-34a调控均不清楚。本项目拟通过体外rMSCs复制性衰老模型和体内rMSCs自然衰老模型，利用基因转染、RNAi技术及双荧光素酶报告基因实验等方法，研究Nampt对rMSCs衰老的作用，进一步探讨miR-34a-Nampt网络在干细胞衰老中的调控机制。我们发现：在rMSCs细胞衰老模型中，miR-34a表达呈现显著的增龄性升高和代次依赖性增加，而Nampt表达则相反。Nampt抑制能诱导rMSCs衰老，而Nampt过表达则能抑制rMSCs衰老，Nampt对rMSCs衰老的调控作用可能是通过介导NAD+合成及Sirt1活性来实现的。miR-34a过表达能促进rMSCs衰老，而miR-34a沉默则能抑制rMSCs衰老，miR-34a对rMSCs衰老具有调控作用，该作用可能是通过直接靶向Nampt进而影响NAD+-Sirt1轴来实现的。本研究从表观遗传学角度揭示miR-34a-Nampt网络调控rMSCs衰老的分子机制，既为研究干细胞衰老的分子机制提供了理论依据和实验数据，也为激活衰老的干细胞，维持干细胞数量及功能，满足种子细胞需求以及防治老龄化疾病提供新策略与新靶点。