LKB1/AMPK信号通路对间充质干细胞衰老的作用机制研究

Aging of mesenchymal stem cells (MSCs) plays a pivotal role in tissue or organism aging, it is important to clarify the signaling pathways and regulatory mechanism associated with the MSCs aging process. Our previous studies have found that gradual increase of liver kinase B1 (LKB1) expression in senescent MSCs, further investigation found it highly expressed in the different microenvironment induction senescence and differentiation of MSCs. Which indicates that LKB1 plays a critical role in MSCs aging process. In this project, we need to test the levels of phosphorylation adenosine monophosphate-activated protein kinase (AMPK). we intended to establish an aging model of MSCs to investigate the association between LKB1/AMPK signaling pathways and senescence-associated factors p16, p21 and p53 of aging MSCs.Using of RNA interference,overexpressed of LKB1,gene chip expression profiles and make clear of the identity of LKB1 as a aging related protein both in vitro. Furthermore, We ought to develop a tandem affinity purification (TAP) methods coupled with mass spectrometry and co-immunoprecipitation to detect the possible interaction proteins of endogenous LKB1.We also wish to investigate the detail molecular mechanismsfor LKB1 in MSCs aging process, analysis its regulation, target substrate and involving signal pathway in vitro.

间充质干细胞（MSCs）的老化是导致组织器官衰老的重要因素，而MSCs衰老的调控机制与某些信号通路改变密切相关。本课题组以往研究表明抑癌基因LKB1伴随MSCs老化进程而逐渐升高，进一步研究发现LKB1与MSCs增殖能力呈负相关，在不同微环境诱导MSCs衰老后LKB1表达明显升高，上述结果提示LKB1可能是MSCs衰老的重要调控分子。因此，本项目拟定重点检测其下游信号靶分子AMPK的磷酸化表达情况；制备MSCs衰老模型，明确LKB1/AMPK信号通路活化与衰老相关基因p16、p21和p53的调控作用；通过RNA干扰和过表达LKB1体系的制备明确其衰老调控蛋白的身份,同时采用基因表达谱芯片分析；拟建立窜联标签标记内源性LKB1，并结合免疫沉淀和质谱分析鉴定其相互作用蛋白。从而深入研究LKB1调控MSCs衰老分子机制，并进一步寻找其他功能活化底物，揭示其在MSCs衰老中的作用机理。

本研究项目主要围绕“LKB1/AMPK 调控间充质干细胞衰老的分子机制”进行实验验证，经过四年的深入研究，目前已充分验证了LKB1/AMPK信号通路参与调控间充质干细胞衰老事件，并发挥了重要作用。我们充分论证了在不同微环境下间充质干细胞增殖、凋亡和衰老进程的变化；同时通过Real-time PCR和Western blot检测年轻和衰老间充质干细胞中LKB1及其下游分子（p53、p21、p16）在mRNA转录水平、蛋白水平表达情况和磷酸化水平；为了验证LKB1/AMPK信号通路的调控机制，我们进一步验证对衰老间充质干细胞生物学功能的影响，即干涉年轻和衰老间充质干细胞的LKB1表达后，其增殖、分化、凋亡、氧自由基水平及β-半乳糖苷酶检测的功能改变。在分子水平进一步验证LKB1干涉后，LKB1/AMPK信号通路下游分子p53、p21、p16表达情况和磷酸化情况，充分证明了衰老间充质干细胞中LKB1/AMPK信号通路活化表达增强，该信号通路活化是MSC衰老进程的关键通路；此外，我们深入研究发现LKB1/AMPK信号通路还可通过调节GSK3参与MSC衰老事件，证明该信号通路可通过多条途径调控间充质干细胞衰老的进程。完成课题后撰写论文7篇，其中已发表3篇中文核心，SCI 3篇，审稿中SCI 1篇。获授权实用新型专利6项，申请中发明专利1项，获贵州省医学科技奖三等奖1项。培养在读硕士研究生4人，在读博士研究生2人，在站博士后2人，晋升副高级职称1人，已达到课题预期成果要求。