LKB1-AMPK激酶在诱导多功能干细胞过程中功能和作用机制的研究

Induced pluripotent stem cells (iPS cells) are the cells of the embryonic stem cell characteristics by somatic cells reprogramming after induction. Induction efficiency of iPS cells, however, needs to be further improved. Studies have shown that AMPK activation is a metabolic barrier to the reprogramming of somatic cells into stem cells. LKB1-AMPK is the key regulator of cellular energy metabolism. The definite mechanisms of whether LKB1-AMPK pathway is involved in the IPS process is unclear. Our preliminary results showed that, in MEF cells, knockout AMPK or LKB1 promoted the expression of c-Myc, and showed the morphological characteristics of the stem cells. Therefore, in this project, we will further confirm the effects of knockout LKB1 or AMPK in the MEFs on reprogramming into iPSCs and detect their stem cell characteristics. The role of AMPK on IPS process and whether AMPK is the only path of the LKB1 affect IPS process will be recognized and cleared by using AMPK agonists and antagonists This project will help to broaden and deepen the understanding of the role of LKB1-AMPK kinase, to improve the study of the mechanism of the IPS process, and to explore new methods to improve IPS efficiency.

诱导多功能干细胞（iPS细胞）是由成体细胞经诱导后重编程表现出胚胎干细胞特性的细胞。然而iPS细胞的诱导效率有待进一步提高。有研究表明，AMPK的激活是成体细胞重编程为干细胞的一个代谢障碍。LKB1-AMPK是细胞能量代谢的关键调节激酶，然而目前对LKB1-AMPK在IPS过程中的作用机制尚不清楚。我们的初步研究结果表明，在MEF细胞中，敲除AMPK或LKB1促进c-Myc的表达，并表现出干细胞的形态特征。因此，在本课题中，我们将进一步证实敲除LKB1或AMPK对MEF重编程为iPSCs的作用及其机制，并检测iPSCs的干细胞特性。并用AMPK激动剂和拮抗剂确认AMPK对IPS过程的作用，明确AMPK是否是LKB1影响IPS过程的唯一通路。本课题有助于拓宽和深化对LKB1-AMPK激酶作用的认识，完善对IPS过程机制的研究，为提高IPS效率探索新方法。

诱导多功能干细胞(iPS细胞)是由成体细胞经诱导后重编程表现出胚胎干细胞特性的细胞，然而iPS细胞的诱导效率有待进一步提高。AMPK的激活是成体细胞重编程为干细胞的一个代谢障碍，而LKB1-AMPK是细胞能量代谢的关键调节激酶。本研究初步探讨LKB1-AMPK在iPS诱导过程中的作用及其可能机制。本项目以小鼠胚胎成纤维细胞株MEF及建立的敲减LKB1的MEF(LKB1 KD)细胞和敲减AMPKα1α2的MEF(AMPKα1α2 KD)细胞为对象，观察敲减LKB1和敲减AMPKα1α2对MEF细胞重编程为干细胞过程的影响,证实敲减LKB1或AMPK对MEF 重编程为iPSCs的作用及其机制,并检测iPSCs的干细胞特性。差异基因筛选结果显示三类细胞差异基因均大于1000个，且AMPKα1α2 KD MEF细胞和LKB1 KD MEF细胞这两者的差异要远大于它们各自与WT MEF细胞的差异。三类细胞经诱导后均能形成干细胞样克隆,但WT细胞形成的干细胞克隆比AMPK KD MEF更大，免疫组化观测到干细胞克隆ALP检测阳性和SSEA、Oct4阳性细胞。实时荧光定量PCR和western blot测定各组细胞均有iPS标志基因SSEA、Oct4、Sox2、Klf4、c-Myc、Nanog 、ALP的表达。本课题有助于拓宽和深化对LKB1-AMPK激酶作用的认识，完善 对IPS过程机制的研究，为提高IPS效率探索新方法。