【作者】 张静；

【导师】 张钲；

【作者基本信息】 兰州大学 ， 内科学·心血管病， 2017， 博士

【摘要】 【背景与目的】线粒体是高动力学细胞器,在细胞多种生命活动中起重要作用。线粒体蛋白酪氨酸磷酸酶1(PTPMT1)是一种新发现的仅存在于线粒体内的PTEN样蛋白酪氨酸磷酸酶,广泛表达于多种组织中,破坏PTPMT1的表达会严重干扰线粒体功能,并引起一系列疾病。骨髓间充质干细胞(MSC)是具有自我更新和多向分化潜能的干细胞,由于MSC具有促进造血及组织修复的能力,使得MSC广泛应用于多种疾病的细胞治疗。然而,我们对于MSC增殖与分化过程中能量代谢及细胞氧化还原状态的变化却知之甚少。因此,本研究旨在明确PTPMT1如何通过影响线粒体的生物生成和底物利用,从而进一步调控MSC的自我更新及分化。【方法】通过基因打靶技术构建PTPMT1完全性敲除或在造血系统中特异性敲除的小鼠,并从小鼠体内纯化培养MSC,检测野生型MSC(WT MSC)和PTPMT1敲除MSC的细胞增殖、凋亡、分化、细胞周期、能量代谢以及细胞内信号转导通路的改变等。【结果】小鼠体内PTPMT1基因完全性敲除导致胚胎发育停滞及着床后死亡。将造血系统中PTPMT1敲除后,小鼠体内干细胞池显著增加。相比于WT MSC,PTPTM1敲除MSC的细胞增殖减慢且向脂肪细胞的分化完全受阻,同时,细胞内周期蛋白依赖性蛋白激酶抑制剂上调以及细胞周期阻滞。进一步的研究表明,PTPMT1敲除MSC的有氧代谢降低,而细胞的糖酵解增加。此外,在应激状态下,PTPMT1敲除的MSC对于丙酮酸的有氧代谢下降,对于氨基酸和脂肪酸的代谢则高于WT MSC。【结论】本研究证实了PTPMT1对于小鼠早期胚胎发育及干细胞的代谢调控具有至关重要的作用。作为线粒体压力介导的检查点,PTPMT1通过调控细胞代谢影响MSC的自我更新与分化。更多还原

【Abstract】 Background and aim: Mitochondria are highly dynamic organelles that play multiple important roles in cells.Protein tyrosine phosphatase mitochondrial 1(PTPMT1),a newly identified PTEN-like protein tyrosine phosphatase,is exclusively localized to the inner membrane of mitochondria,and widely expressed in various tissues.Disruption of PTPMT1 expression severely interferes with the function of mitochondrial,leading to a serious disease.Mesenchymal stem cell(MSC)is multipotent stem cell,which has clonal self-renewal and multilineage differentiation potential.Given the innate ability of these cells to promote hematopoiesis recovery and tissue repair,there is rising interest in utilizing MSC in a broad repertoire of cell based therapies for the treatment of diseases.However,the regulation and coordination of mitochondrial metabolism with MSC proliferation and differentiation is not fully understood.Here,we focus on the mechanism how mitochondrial energy metabolism is regulated and how mitochondrial bioenergetics and substrate utilization cooperatively coordinate both MSC self-renewal and differentiation.Methods: we created both Ptpmt1 null and conditional(floxed)alleles by gene targeting and generated Ptpmt1-deficient animal and cell models.We determined the effects of PTPMT1 depletion on MSC by analyzing cell proliferation,apoptosis,differentiation,cell cycle,energy metabolism and cell signaling.Results: Global disruption of PTPMT1 resulted in developmental arrest and postimplantation lethality.Deletion of PTPMT1 in hematopoietic-cell-specific knockout mice resulted in the stem cell pool drastically expanded.Deletion of PTPMT1 in MSC decreased proliferation and essentially blocked the adipocyte differentiation.This was accompanied by upregulation of cyclin-dependent kinase inhibitors and a significant cell cycle delay.Further analysis demonstrated that oxygen consumption of Ptpmt1-depleted cells was decreased,while glycolysis was concomitantly enhanced.In addition,under stress condition,compared to WT MSC,the aerobic metabolism of pyruvate in PTPMT1 knockout MSC decreased while the amino acid and fatty acid metabolism increased.Conclusion: This study established a crucial role of PTPMT1 in embryogenesis of mice and metabolic regulation of stem cell function.As a mitochondrial metabolic stressactivated checkpoint,PTPMT1 controlls MSC self-renewal and differentiation by metabolic regulation.更多还原