AMPK在诱导性多潜能干细胞线粒体重编程中的调节作用

Under physiological conditions, glucose oxidation via mitochondrial oxidative phosphorylation is the major source of energy (ATP) for somatic cells, while glycolysis represents a supplementary approach in coping with stresses. Although ATP-generating efficiency of glycolysis is much lower than that of oxidative phosphorylation, tumor cells acquire aerobic glycolysis as the major source of energy, a phenomenon called Warburg effect and a hallmark of tumor cell metabolism. Recent studies have demonstrated that induced pluripotent stem cells (iPS) exhibit the same trait which is described as mitochondria reprogramming, an event that precedes and is required for nuclear reprogramming. The tumor suppressor AMP-activated activated protein kinase (AMPK) has been shown to inhibit the Warburg effect, possibly through which it inhibits the induction of pluripotent stem cells. Our preliminary data have demonstrated that AMPK is located at mitochondria. Based on previous studies and our preliminary findings, we hypothesize that mitochondria-associated AMPK promotes oxidative phosphorylation, thereby inhibiting mitochondria reprogramming and the induction of pluripotent stem cells. Therefore, to test this hypothesis, we will: (1) examine changes in AMPK activity and mitochondria localization during the course of stem cell induction, (2) test if AMPK regulates mitochondria reprogramming and, (3) explore if inhibition of AMPK enhances the efficiency of stem cell induction. Completion of this study will be instrumental to understanding the mechanism underlying AMPK inhibition of iPS reprograming and provide an important approach to increase the efficiency of pluripotent stem cell induction.

正常体细胞通过线粒体葡萄糖氧化磷酸化获取能量，在应急状态下，细胞质糖酵解用以补充能量，虽然它的产能效率远低于氧化磷酸化，肿瘤细胞利用它作为主要产能途径，这一现象被称为Warburg效应。新近的研究表明，这一特征性转变也存在于诱导性多潜能干细胞中，被谓之为线粒体的重编程序。它先于干细胞诱导过程中的细胞核重编程序，为干细胞诱导的先决条件。有研究显示，AMPK可以抑制Warburg效应，并有可能藉此抑制多潜能干细胞的诱导。我们的前期工作显示AMPK存在于细胞线粒体上。综合上述，我们假设，线粒体AMPK促进细胞氧化磷酸化，以此抑制线粒体的重编程序。因此，我们将检测在多潜能干细胞诱导过程中AMPK活性和在线粒体的定位变化；研究线粒体AMPK活性的改变对干细胞诱导的影响；通过下调AMPK来提高干细胞的诱导效率。该研究有助于阐述AMPK抑制多潜能干细胞的诱导机制，开拓增加诱导效率的新途径。

近年来，研究表明在干细胞因子重编程体细胞的过程中，线粒体也发生重编程，其数量、结构和功能发生相应改变，且先于干细胞诱导过程中的细胞核重编程，是干细胞诱导的先决条件。单磷酸腺苷活化蛋白激酶(AMPK)是生物能量代谢调节的关键分子，在正常细胞和肿瘤细胞中均发挥重要的生物功能。AMPK对能量代谢的调节与线粒体的功能密不可分，为此本课题着重探究了AMPK对线粒体的调控作用：(1)通过免疫荧光和生物化学手段证明AMPK定位于线粒体内，且AMPKβ亚基与β-ATP-synthase存在共定位。(2)通过构建AMPK活性突变体腺病毒、AMPKβ亚基敲除细胞株分析了AMPK对线粒体数量、生物合成、分裂与融合的调控作用。研究发现，当AMPK激活时，线粒体DNA和蛋白含量显著增加，上调线粒体生物合成关键蛋白PGC1α和Nrf-1的表达，上调动力相关蛋白Drp-1的磷酸化水平，促进线粒体分裂。而敲除AMPKβ亚基，线粒体DNA和蛋白含量显著降低，下调生物合成PGC1α和Nrf-1的表达，上调线粒体融合蛋白Mfn1/2的表达，促进线粒体的融合，而表达AMPKα持续活性突变体部分逆转β亚基敲除的效应。(3)观察能量代谢改变发现，特异性靶向线粒体的AMPK持续活性突变体会抑制糖酵解，促进脂肪酸氧化，而AMPKβ1和AMPKβ2亚基敲除促进糖酵解。(4)AMPK激活剂二甲双胍抑制干细胞转录因子Oct4、Nanog和c-Myc的表达，抑制肿瘤干细胞形成。本研究有助于阐明AMPK对线粒体重编程的调控机制，为提高干细胞诱导效率和抑制肿瘤干细胞提供新途径和策略。