【作者】 汪敏慧；

【导师】 曹励之；

【作者基本信息】 中南大学 ， 儿科， 2009， 硕士

【摘要】 研究背景:化疗是目前儿童白血病治疗的主要手段,白血病化疗失败的主要原因是白血病的多药耐药(multidrugresistance,MDR),尤其对于儿童急性非淋巴细胞白血病(acutenonlymphoblastic leukemia,ANLL),多药耐药的发生严重制约着其长期无事件生存率的进一步提高。前期研究已经证实了细胞骨架调节蛋白WASP家族富含脯氨酸同源蛋白1(WASP-family verprolinhomologous protein 1,WAVE1)特异性参与了儿童急淋的发病,与儿童急淋病程呈正相关,表现为活动期(初发和复发患者)WAVE1的表达持续增高,完全缓解期表达恢复到正常水平,并初步发现了WAVE1参与了白血病多药耐药的形成。进一步明确WAVE1对多药耐药相关基因表达的影响,为白血病多药耐药的逆转提供一个新的思路。目的:探讨儿童ANLL中WAVE1基因参与多药耐药基因(multidrug resistance 1,MDR1)、多药耐药相关蛋白基因(multidrugresistance associated protein 1,MRP)、乳腺癌耐药蛋白基因(Breastcancer resistance protein,BCRP)表达和调控的机制。方法:应用实时荧光定量PCR(RQ-PCR)法分别检测52例ANLL患儿治疗前及疾病发展过程中骨髓单个核细胞(bone marrowmononuclear cells,BMMCs)中WAVE1,MDR1,MRP1及BCRP mRNA的表达水平。将pCDNA3.1A-WAVE1真核表达质粒转染K562细胞构建WAVE1高表达K562细胞,用RQ-PCR法检测转染前后白血病细胞系WAVE1,MDR1,MRP1及BCRP基因的表达水平。结果:52例入组患儿中共有21例复发或初次诱导未缓解,31例处于持续缓解状态,复发难治组的WAVE1及耐药相关基因表达水平明显高于缓解组患儿(P＜0.05),同一患儿缓解期WAVE1的表达水平明显低于初治期及复发期(P＜0.05);与K562相比较,K562/A02中WAVE1及MDR1的mRNA表达水平均明显增加;转染WAVE1质粒后的K562中MDR1、MRP1、BCRP的mRNA表达水平均有不同程度的增高。结论:WAVE1与儿童ANLL的病程及预后相关,WAVE1参与了髓系白血病细胞多药耐药的形成并可影响多个重要的耐药相关基因的表达。更多还原

【Abstract】 Background: Chemotherapy is one of the main treaments for children with acute leukemia (AL). Multidrug resistance (MDR) is an important reason of treatment failure in children with acute leukemia, especially in acute nonlymphoblastic leukemia (ANLL). The occurrence of multidrug resistance strongly decreases the long-term event-free survival rate.In previous researches, we have confirmed that cytoskeletal regulatory protein WASP-family verprolin homologous protein 1(WAVE1) is involved in the pathogenesis of children with acute lymphoblastic leukemia (ALL), and with positive impact. At the active stage (in patients with primary and recurrent), the expression of WAVE1 continued to increase, while back to normal levels at complete remission stage. WAVE1 was also found involved in MDR. It can be a new way to reverse multidrug resistance if we can confirm WAVE1’s impact to multidrug resistance genes.Objective: To investigate WAVE1’s impact to the expression and regulation of multidrug resistance gene 1, multidrug resistance-associated protein gene 1, and breast cancer resistance protein gene.Methods: WAVE1, MDR1, MRP1, BCRP mRNA expression in bone marrow mononuclear cells (BMMCs) were tested by Real-time fluorescence quantitative PCR (RQ-PCR) in a cohort of fifty-two children with acute myeloblastic leukemia. K562 cell was transient-transfected by PCDNA3. 1A-WAVE1 reconstructed plasmid and the expression of WAVE1, MDR1, MRP1 and BCRP before and after transfection were investigated by real-time PCR.Results: Twenty-one patients were refractory or relapsed during the follow-up and thirty-one patients were found in continuous remission. Levels of WAVE1, MDR1, MRP1, BCRP’s expression in refractory/relapsing group were much higher than in complete continuous remission (CCR) group (P<0.05). Compared with continuous remission group we showed that higher expression of WAVE1 mRNA in newly diagnosed or relapsed ANLL children’s BMMCs (P<0.05).WAVE1 and MDR1 mRNA expression in K562/A02 was much higher than that in K562.Levels of WAVE1, MDR1, MRP1, BCRP mRNA expression were increased to varying degrees after transfecting pCDNA3. 1A-WAVE1 reconstructed plasmid in K562 cells.Conclusion: Our results suggested that higher levels of WAVE1 in BM indicates an unfavorable prognosis in children with ANLL. WAVE1 is involved in the MDR mechanisms and regulates the level of MRP1 and BCRP expression.更多还原