HO-1基因调控在急性髓系白血病治疗中的作用及机制研究

Presently the treatment of acute myeloid leukemia (AML) is largely dependent on chemotherapy and hemopoietic stem cell transplantation. Due to the accumulating toxic effect of chemicals, high ratio of relapse, strict donor-selection criteria and severe complications, the disease-free survival and the total survival rate are generally dissatisfying, which promotes the research on target-oriented therapy in the treatment of AML. The correlation of HO-1 gene with tumor has been recently reported. Our previous experiments have demonstrated that in chronic myelocytic leukemia and AML HO-1 is overexpressed which is related with drug-resistance. In the present study, recombinant lentivirus for HO-1 gene and RNA interference are constructed and transferred into target cells, and gene regulation targeting the endogenous genes is performed using revulsants and inhibitants. Real-time PCR, FISH, flowcytometry, Transwell test, gene sequencing plus animal experiments are carried out to study the association of HO-1 expression with the risk stratification, drug resistance in acute myeloid leukemia and to investigate the related signal pathways. Leukemic stem cells are transfected with target gene to elucidate the effects of HO-1 regulation on the apoptosis, proliferation and drug resistance of leukemic stem cells, to manifest the role of HO-1 as a potentially therapeutic target and to provide theoretical and experimental evidence for AML target therapy.

急性髓系白血病（AML）目前的治疗手段主要是化疗和造血干细胞移植，但化疗的毒性和高复发率及移植供受者的严格选择和严重并发症，使无病生存率和总生存率难以提高，因此寻找AML潜在治疗靶点，探索靶向治疗成为研究热点。HO-1基因与肿瘤的相关性近期已有报道，本课题组前期实验证实了HO-1在CML、AML中的高表达，并提示了与抗凋亡和耐药相关。本研究构建含HO-1的重组慢病毒载体和HO-1的沉默RNA转染靶细胞，应用诱导剂和抑制剂对内源性HO-1进行基因调控，采用real-time PCR、FISH、流式细胞术、Transwell实验、基因测序和动物实验，研究HO-1基因表达与AML的危险分层和耐药的关系，阐明信号通路，以白血病干细胞(LSC)作为转基因靶细胞，研究HO-1的表达调控和LSC的凋亡、增殖、复发和耐药等关系，试图证实HO-1可作为AML的潜在治疗靶点，为AML的靶向治疗提供实验依据。

化疗的毒性和高复发率及移植供受者的严格选择和严重并发症是急性髓系白血病（AML）的主要瓶颈。寻找AML潜在治疗靶点，探索靶向治疗成为研究热点。HO-1基因已被证实与多种细胞增殖及肿瘤耐药相关。本课题组对HO-1在AML恶化，AML疾病危险分层，慢性粒细胞白血病（CML）转AML中的作用进行了深入的研究。其相关内容包括：.（1）在急性髓系白血病细胞中，HO-1高表达与DNA修复机制关键因子INPP4B的表达呈正相关。HO-1可以促进INPP4B在AML细胞KG-1中的表达，从而降低p-H2AX。敲除敲低INPP4B能够显著下调DNA修复通路的关键因子ATM的表达。.（2）HO-1高表达可以激活JNK/c-Jun信号转导通路抑制AML细胞的凋亡并延长AML模型小鼠的生存率。AML-M5的患者中HO-1表达明显高于其他分型，小干扰RNA沉默U937细胞中HO-1表达，可以增加其对阿糖胞苷的敏感性。动物实验发现感染了siRNA-HO-1的小鼠模型，肿瘤生长情况远不如未感染组。.（3） 证实HO-1，RET和PML可能是AML预后较差的标识。将90例AML患者分为低危，中危，高危三组，在基因水平上检测HO-1，基因表达。发现随着患者预后从低危到高危，HO-1表达逐渐升高。.（4）在急性白血病中，HO-1可以作为造血干细胞移植的预示标识。当HO-1表达升高可能预示急性白血病复发，而在移植后，HO-1在骨髓中的高表达则预示着可能发生aGVHD。.（5）低氧的骨髓微环境能够诱导白血病干细胞中HIF-1a的过表达，该因子是AML患者预后较差的标识。我们采用2ME2抑制HIF-a表达，可以增加AML对阿糖胞苷的敏感性。同时，可以下调下游VEGF,GLUT1和HO-1的表达。.（6）证实细胞钠-氢离子泵通过激活PKC-βHO-1信号转导通路促使CML发生耐药，从而发展到加速期及急变期。IM耐药的CML细胞中pH值升高可上调HO-1，从而对伊马替尼耐药。 .（7）证实HO-1可以通过抑制mTOR信号转导通路诱导自噬的发生，从而导致CML对伊马替尼耐药。沉默HO-1在K562R细胞中的表达时，自噬现象减弱，同时细胞对伊马替尼敏感度增加。.　　　综上所述，HO-1是治疗AML的新靶点，抑制HO-1在AML细胞中的表达，可以显著增加AML细胞对化疗药物的敏感性。