自噬活性与白血病细胞获得性耐药的关系研究

Autophagy is an evolutionarily conserved and lysosome dependent protein degradation pathway. Our previous study indicated that macroautophagy and CMA were activated sequentially instead of simultaneously in starvation or chemotherapy drug-induced cancer cells in which damaged or superfluous proteins and organelles were cleared in different ways, the aim is to realize the recycling of material and help cells to endure the current crisis. From which we can infer that macroautophagy and CMA may be highly correlated to the mechanism of tumor resistance to chemotherapy. In this projects, We will transfer the green fluorescent protein (GFP) gene into K562 cells and establish K562-GFP cell line which the GFP was persistently expressed. The GFP labeled leukemia cells will be induced by adriamycin to establish a stable drug-resistance cell line (K562-GFP/ADM), then, in vitro and in vivo, dynamically observe the change trend of autophagic activity in different stages of drug resistance. Furthermore, we will artificially change the autophagy level to investigate whether the autophagy is involved in the molecular mechanisms for acquired chemoresistance in leukemia cells. This study will be helpful for explore the therapy strategy of inverting drug resistance, it will plays an important role in clinical treatment of resistant and relapsed leukemia.chemoresistance in leukemia cells and its LSCs, and the number and other features of LSCs. This study will be helpful for explore the therapy strategy of inverting drug resistance and eliminate LSCs, it will plays an important role in clinical treatment of resistant and relapsed leukemia.

自噬是进化上高度保守的溶酶体降解途径。我们的前期研究表明当肿瘤细胞受到营养缺乏和化疗药物等压力刺激时，巨自噬与分子伴侣介导的自噬（CMA）相继顺序而非同步被激活，以不同方式降解受损的细胞器和蛋白质等内容物，以实现物质的循环再利用，帮助细胞度过危机。由此我们推断，自噬（巨自噬和CMA）可能是肿瘤细胞对抗癌药物产生抗性及抗性维持的重要机制。本课题将绿色荧光蛋白（GFP）基因转入白血病K562细胞中建立能稳定表达GFP的K562-GFP细胞株，并通过ADM长期间歇性诱导建立具有稳定耐药性的K562-GFP/ADM细胞株，体内外动态观察不同耐药阶段细胞自噬活性的变化，并通过化学药物或慢病毒载体shRNAs沉默自噬相关基因改变细胞自噬水平，研究自噬活性与白血病细胞获得性耐药及其特性维持之间的关系及调控机制。探索通过抑制细胞自噬活性来逆转白血病耐药，在临床耐药性和复发性白血病治疗中发挥重要作用。

白血病是造血系统的恶性肿瘤,化疗依然是治疗白血病的主要方法，但化疗过程中药物诱导产生的耐药现象是临床化疗失败的主要原因之一。本课题将绿色荧光蛋白（GFP）基因转入白血病K562细胞中建立能稳定表达GFP的K562- GFP细胞株为研究对象，用蒽环类化疗药物阿霉素（adriamycin，ADM）体外“逐步提高药物浓度+间歇性诱导刺激”的方式逐步诱导建立对ADM有稳定耐药性的K562- GFP /ADM细胞。一方面我们通过观察K562- GFP /ADM细胞对柔和霉素、As2O3、吡柔比星、5-FU、长春新碱等其它化疗的敏感性，发现K562- GFP /ADM细胞除了对ADM产生显著耐药外，还对多种化疗药物如：吡柔比星、柔红霉素、5-FU和长春新碱等有交叉耐药，但对三氧化二砷较敏感，从而筛选出与常规化疗药物不存在交叉耐药的新型药物，为临床攻克白血病多药耐药提供研究基础。另一方面我们通过观察K562- GFP /ADM细胞耐药前后细胞自噬水平的变化，并用自噬抑制剂3-甲基腺嘌呤（3-methyladenine，3-MA）抑制细胞自噬，观察自噬水平改变与K562- GFP /ADM细胞化疗敏感性及耐药相关基因表达水平的关系，我们发现K562- GFP /ADM细胞内自噬体数量、MDC荧光强度以及LC3Ⅰ/Ⅱ、Beclin-1等自噬相关蛋白表达水平均显著高于亲本细胞。用3-MA抑制自噬可明显增加K562/ADM细胞对ADM的敏感性，同时也能有效抑制K562/ADM细胞内耐药相关蛋白的表达。此外，我们在用ADM诱导K562- GFP细胞耐药过程中，对不同耐药程度的K562- GFP /ADM细胞的自噬活性进行动态检测，发现K562- GFP /ADM耐药程度与细胞自噬水平呈正相关。本课题的研究结果表明K562- GFP /ADM细胞获得性耐药与细胞自噬水平有密切关系，自噬活化是导致白血病细胞获得性耐药的重要机制。