【作者】 黄莹；

【导师】 陈国强；

【作者基本信息】 中国科学院研究生院（上海生命科学研究院） ， 生物化学与分子生物学， 2006， 博士

【摘要】 磷脂爬行酶1(phospholipid scramblase 1,PLSCR1)属于钙离子结合的、棕榈酰化II型膜蛋白,但最近的研究显示非棕榈酰化PLSCR1也能进入细胞核内并结合基因组DNA。目前,有关PLSCR1的生物学功能尚不完全清楚。在过去的工作中,本实验室报告蛋白激酶Cδ(PKCδ)通过序列活化JNK和STAT1信号分子调控PLSCR1的表达。在此基础上,我们发现PLSCR1在全反式维甲酸(ATRA)、佛波酯(PMA)诱导的白血病细胞分化过程中发挥作用。基于这些发现,本课题试图在利用四环素关闭的基因表达系统建立诱导表达PLSCR1的髓系白血病细胞系的基础上,研究PLSCR1对白血病细胞的生物学行为的影响,并初步探讨其分子机制。本文主要取得如下结果:(1)利用四环素-关闭基因表达系统建立了诱导表达PLSCR1的髓系白血病细胞系U937plscr1,并确定了诱导表达的PLSCR1主要分布在U937细胞的胞膜及胞浆中,少量分布于细胞核中;(2) PLSCR1的诱导表达能有效抑制白血病细胞U937的增殖、伴随细胞周期阻滞在G1期,并促使其朝向粒系分化。与此同时,p27、p21、SKP2和c-Myc等多种细胞周期及分化相关蛋白的表达也发生改变;(3)诱导表达的PLSCR1在下调抗凋亡蛋白bcl-2表达的同时,增强对化疗药etoposide引起的细胞凋亡的敏感性,表现为PKCδ、caspase-3的活化、线粒体跨膜电位的崩塌、凋亡核型的改变以及PARP剪切等显著加强.综上所述,本文提供了PLSCR1在白血病细胞分化中发挥作用的直接证据,并提出PLSCR1可以发挥抗白血病作用的观点。这些工作拓展了人们对于PLSCR1生物学功能的认识,对于认识白血病细胞分化的分子机制,进而为识别诱导分化治疗白血病的药物靶标提供了重要理论基础。更多还原

【Abstract】 Phospholipid scramblase 1 (PLSCR1) is a multiply palmitoylated, calcium-binding type II transmembrane protein, while unpalmitoylated PLSCR1 was also found to be localized into the nucleus where it binds to genomic DNA. However, the exact mechanisms of expression regulation of PLSCR1 gene and its biological functions remain largely unclear. More recently, our laboratory found that protein kinase Cδ(PKCδ) upregulates PLSCR1 expression via the sequential activation of c-Jun N-terminal kinase (JNK) and singnal transducer and activator of transcription 1 (STAT1). What’s more, PLSCR1 acts a role in the all-trans retinoic acid (ATRA) and phorbol 12-myristate 13-acetate (PMA) -induced leukemic cell differentiation. In the present work, we try to establish an inducible PLSCR1-expressing myeloid leukemic cell line to explore direct evidence of potential roles of PLSCR1 in the leukemic cell differentiation. Towards this end, the following original and interesting results were gotten:(1) An inducible human PLSCR1-expressing leukemic cell line U937plscr1 was generated using tet-off gene inducible system and overexpressing PLSCR1 protein was found to be distributed largely in the plasma membrane and cytoplasm with a little in the nucleus.(2) Upon PLSCR1 induction, the proliferation of U937plscr1 cells was arrested at G1 phase of the cell cycle. More importantly, PLSCR1 overexpression alone could also drive leukemic U937 cells to undergo differentiation towards granulocyte-like cells, as determined by morphology, differentiation markers and differentiation-related gene expressions. In aggrement with changes of these cellular behaviors, PLSCR1 expression also increased p27Kip1/p21Cip1 proteins and reduced c-Myc, SKP2 proteins, which induce growth arrest and cell differentiation.(3) PLSCR1 induction increased cellular sensitivity to etoposide-induced apoptosis with decreased Bcl-2 protein and enhanced proteolytic cleavage of PKCδ, the ?Ψm collapse, caspase-3 activation, apoptotic nuclear changes, and PARP cleavage as well.Taken together, this work proposes that PLSCR1 exerts anti-leukemic effects through inducing growth arrest and differentiation as well as increasing sensitivity to apoptosis induction. These findings shed new insights for understanding the biological functions of PLSCR1 and provide the theoretic basis for further exploring the molecular mechanisms of leukemic cell differentiation and for recognizing new drug targets of differentiation therapy.更多还原