p19INK4d调控慢性粒细胞白血病红系分化作用及机制研究

Chronic myeloid leukemia (CML) is a serious hematologic malignancy. differentiation arrest of hematopoietic stem or progenitor cells plays a pivotal role in the occurrence of CML. p19INK4d, an important cyclin dependent kinase inhibitor, is closely related to the pathogenesis of many diseases, moreover, plays an important regulatory role in the human erythroid terminal differentiation. Our previous results showed that p19INK4d expression was significantly reduced in CML patients. Overexpression of p19INK4d inhibited K562 cells proliferation and enhanced inhibitory effect of imatinib on K562 cells, furthermore, the expression of γ-globin and glycoprotein A (GPA) was significantly increased, however, the detailed mechanism is still unclear. Western blot results indicated that p19INK4d up-regulated expression of Sox6 gene which was involved in erythroid differentiation. In this study, we will further elucidate that p19INK4d up-regulates the expression of γ-globin and GPA by regulating Sox6 gene, and induced erythroid differentiation of CML cells through clinic, molecular, cell and animal level. Meanwhile, our investigation contributes to reveal the molecular mechanism that p19INK4d enhances the efficacy of imatinib through inducing erythroid differentiation，which provides new strategy for clinical diagnosis and treatment of CML patients.

慢性粒细胞白血病（CML）是一种危害严重的恶性血液系统肿瘤，造血干/祖细胞分化成熟阻滞是CML疾病发生的关键因素。p19INK4d作为一个重要的细胞周期蛋白依赖性激酶抑制分子，与多种疾病的发生密切相关，在人正常红系终末分化过程发挥重要调控作用。本研究前期结果发现在CML患者中p19INK4d表达明显降低；过表达p19INK4d后K562细胞增殖减慢、增强伊马替尼对K562细胞生长抑制，血红蛋白γ-globin和GPA表达显著升高，但其机制仍不清楚；Western blot结果显示过表达p19INK4d后明显上调红系转录因子Sox6的表达。本研究拟进一步从临床样本、细胞分子、动物实验水平阐明p19INK4d如何通过调控Sox6基因，上调HBG和GPA表达，诱导CML细胞红系分化作用机制；明确p19INK4d诱导红系分化协同提高伊马替尼疗效的机制，为临床上CML患者的诊治提供新策略。

慢性粒细胞白血病（chronic myeloid leukemia，CML）是一种常见且危害很大的造血干细胞恶性克隆增生性疾病。BCR-ABL融合基因与CML发病直接相关，但是越来越多的患者出现复发或耐药现象，因此，寻找新的治疗靶标对突破CML治疗“瓶颈”具有非常重要的意义。研究发现p19INK4d能够特异性抑制CDK4/6的激酶活性调控细胞增殖，然而其与CML分化调控作用机制仍不明确。因此，目前寻找新的肿瘤细胞分化调控分子靶点与信号通路，研究分化治疗的新方法和新手段，是现阶段白血病分化治疗药物研究的热点和难点问题。.本研究发现p19INK4d在CML病人骨髓样本中mRNA和蛋白水平表达均明显降低。p19INK4d过表达（p19INK4d-OE）后CCK-8和软琼脂克隆形成实验均显示CML细胞增殖能力明显抑制和克隆数量大幅减少，CML细胞周期阻滞；p19INK4d-OE促进K562细胞的红系分化，p19INK4d-OE在CML初诊病人骨髓CD34+细胞中也能促进细胞内HBG、GPA的表达从而诱导干细胞向红系分化，调控红系分化重要的关键基因GATA1、KLF1、LSD-1、Ikaros等表达变化不明显，然而，红系分红重要的转录调控因子Sox6增高，细胞内γ-globin生成增多；此外，p19INK4d-OE和伊马替尼两者联合组能更明显降低MDR1、BCR、BCR-ABL表达以及其下游靶基因p-Mek1/2表达，能协同抑制CML细胞增殖；p19INK4d-OE也能促进CML CD34+细胞HBG的RNA水平增高，诱导细胞向红系分化。.综上所述，本研究首次发现了p19INK4d在CML患者中表达降低，且过表达p19INK4d能抑制CML细胞增殖和诱导红系分化，且其与伊马替尼对CML细胞具有协同作用，因此，本研究为突破当前 CML 临床治疗“瓶颈”的提供了重要潜在靶基因。