【作者】 肖湘；

【导师】 张广森；

【作者基本信息】 中南大学 ， 内科学， 2013， 博士

【摘要】 研究背景急性髓细胞白血病(acute myelogenous leukemia, AML)是成年人最常见的急性白血病,目前它的治疗仍以化疗为主,但有70%左右获得缓解的患者最终复发并演变为难治性白血病,导致治疗失败而死亡。临床上导致白血病化疗失败最重要的原因是白血病细胞耐药。有关白血病细胞耐药的机制至今仍未完全阐明,因此寻找新的耐药分子靶标,对于提高急性髓细胞白血病的疗效,改善其预后具有重要的临床意义。能量代谢是肿瘤细胞代谢的中心环节,而线粒体成为近年来研究的热点。分析特定类型肿瘤中线粒体相关基因和蛋白的变化并寻找其规律或许能为肿瘤患者的诊断和治疗提供有意义的线索。线粒体ATP合酶是线粒体氧化磷酸化的限速酶,其p亚单位(ATPsyn-β)是ATP合酶中唯一起催化作用的亚基。有研究者观察到在结肠癌、肺癌、乳腺癌、食道癌、胃癌、肾癌等实体肿瘤细胞中线粒体ATPsyn-β表达下降,并且可能与肿瘤细胞的化疗耐药有关。目前有关急性白血病线粒体ATP酶的研究尚少。我们课题组在前期研究中对慢性粒细胞白血病(chronic myelogenous leukemia, CML)原代耐药细胞及耐药细胞株ATPsyn-β的表达研究显示ATPsyn-β可作为一个独立的耐药相关蛋白参与调节CML急变患者的耐药。本研究旨在观察急性髓细胞白血病患者白血病细胞中线粒体ATPsyn-β的表达及ATP酶活性的变化,并分析其与临床疗效、化疗耐药的关系,为急性白血病耐药机制研究提供新的思路。第一章线粒体ATPsyn-β在急性髓系白血病患者骨髓单个核细胞及CD34+白血病细胞中的表达目的：检测线粒体ATPsyn-β在不同急性髓系白血病患者中的表达,并分析其与AML患者耐药的关系。方法：1.收集我院2010年10月～2012年6月期间急性髓系白血病(非M3型)住院患者骨髓标本118例(包括初诊患者38例,完全缓解患者38例,复发/难治患者42例),骨髓相对正常的非恶性血液病门诊患者骨髓标本20例作为对照。采用荧光定量PCR、western印迹和流式细胞术检测患者骨髓单个核细胞(BMMCs)中线粒体ATPsyn-p mRNA和蛋白的表达水平。2.用CD34正选免疫磁珠分选20例急性髓系白血病患者BMMCs,得到CD34阳性的白血病干/祖细胞,应用荧光定量PCR、 western印迹测定其ATPsyn-β水平。3.用MTT法检测急性髓系白血病患者(缓解患者38例,复发/难治患者42例)BMMCs体外对阿霉素的敏感性,并分析线粒体ATPsyn-β表达水平与体外阿霉素耐药性的关系。结果：1.非M3型急性髓系白血病患者(n=118) BMMCs中线粒体ATPsyn-β mRNA和蛋白水平较正常骨髓BMMCs呈显著性降低,P<0.01。2.与AML缓解组(n=38)相比,复发难治组(n=42)骨髓BMMCs及CD34+细胞线粒体ATPsyn-β mRNA及蛋白水平均下降,P=0.004；且两组患者CD34+细胞中ATPsyn-β mRNA差异较BMMCs来源标本更显著,P=0.001。3.阿霉素对复发/难治组和缓解组AML患者骨髓单个核细胞的IC50分别为13.690μM和1.549μM,前者为后者的8.84倍。4.复发/难治组AML患者线粒体ATPsyn-β mRNA和蛋白表达水平与体外阿霉素对骨髓单个核细胞的IC50值呈显著负相关,P<0.05。结论：1.线粒体ATP合酶p亚单位(ATPsyn-β) mRNA及蛋白质表达在非M3型急性髓系白血病患者中较正常人明显降低。2.复发/难治急性髓系白血病患者骨髓单个核细胞中线粒体ATPsyn-β mRNA及蛋白表达较缓解期患者下调。复发/难治急性髓系白血病患者CD34阳性细胞中ATPsyn-β表达低于缓解期患者。ATPsyn-β表达下调与骨髓单个核细胞体外对阿霉素的耐药性存在负相关,提示ATPsyn-β与化疗耐药有关。第二章难治/复发急性髓系白血病原代细胞及耐药白血病细胞株线粒体ATP酶活性变化及意义目的：测定不同急性髓系白血病患者骨髓单个核细胞中线粒体ATP酶活性,并分析其临床意义。方法：收集42例急性髓系白血病(非M3型)患者骨髓标本并分离单个核细胞,提取BMMCs以及阿霉素耐药细胞株(HL-60/ADM, K562/A02)的线粒体；利用ATP酶水解ATP生成ADP和Pi的原理,用酶标方法测定Pi浓度,以间接反映线粒体ATP酶的活性。结果：1、阿霉素耐药细胞株HL-60/ADM. K562/A02的ATP酶活性(38.22±2.00U,36.70±7.47U)较其亲本细胞株HL-60(132.06±14.65U)、 K562(65.43±1.31U)呈显著降低,P<0.05。2、复发/难治急性髓系白血病患者(n=26) BMMCs线粒体ATP酶平均活性为20.33U,低于缓解组患者(n=16)的31.17U,P<0.05。ATP酶活性的高低与ATPsyn-β mRNA的表达量呈正相关。结论：1.阿霉素耐药白血病细胞株HL-60/ADM、K562/A02存在ATP酶活性显著下降；复发难治急性髓系白血病患者骨髓单个核细胞线粒体ATP酶活性显著地低于缓解组病人,提示线粒体ATP酶活性下降与急性髓系白血病化疗耐药相关。2.线粒体ATP酶活性与ATPsyn-β mRNA的表达水平呈正相关。第三章线粒体ATPsyn-β在急性髓系白血病耐药细胞株中的表达及对细胞增殖、凋亡和耐药特性的影响目的：探讨ATPsyn-β对阿霉素诱导细胞增殖凋亡的影响,通过基因转染、RNA干扰技术分析ATPsyn-β表达水平的变化与HL-60/ADM细胞耐药习性产生及耐药逆转的关系。方法：1.采用荧光定量PCR、western印迹、流式细胞术检测HL-60/ADM耐药细胞株线粒体ATPsyn-β表达。2.用RNA干扰技术抑制ATPsyn-β表达后,MTT法检测干扰前后阿霉素对HL-60细胞的增殖抑制作用；通过瑞氏-吉姆萨染色和Hoechst33258染色观察细胞形态的变化；流式细胞术检测细胞对阿霉素的摄取以及细胞早期凋亡率。荧光定量PCR测定MRP的变化。3.ATPsyn-β重组质粒转染入HL-60/ADM细胞株,MTT法检测转染前后阿霉素对HL-60/ADM细胞的增殖抑制作用；通过瑞氏-吉姆萨染色和Hoechst33258染色观察细胞形态的变化；流式细胞术检测细胞对阿霉素的摄取以及细胞早期凋亡率。荧光定量PCR测定MRPmRNA的变化。结果：1.HL-60/ADM耐药细胞存在ATPsyn-β mRNA水平显著降低,与亲本非耐药HL-60细胞相比,其mRNA水平下降了2.6倍,P<0.01。HL-60/ADM细胞MRP mRNA表达水平较其亲本的HL-60细胞增加了37倍。2.HL-60/ADM耐药细胞线粒体ATPsyn-β蛋白表达水平较其亲本HL-60细胞明显下调,P<0.05。3.在RNA干扰实验,阿霉素对ATPsyn-β干扰组细胞的IC50为1.67±0.241μM,而未干扰组细胞的IC5o为0.37±0.25μM,P<0.05。在重组质粒转染实验,不论阿霉素处理细胞24小时还是48小时,与对照组细胞相比,ATPsyn-β转染组ICso均显著降低,有统计学差异。4.ATPsyn-β-siRNA可抑制HL-60细胞对阿霉素的摄取。阿霉素作用细胞1小时后,与对照相比,同一时间点RNA干扰后的细胞摄取阿霉素荧光阳性率有显著下降。而ATPsyn-β重组质粒可促进HL-60/ADM细胞对阿霉素的摄取,阿霉素作用细胞4小时后,与对照相比,同一时间点转染后的细胞内阿霉素荧光阳性率明显增加。5.RNA干扰下调ATPsyn-β表达可抑制阿霉素诱导的HL-60细胞早期凋亡,而基因转染上调ATPsyn-β表达可促进阿霉素诱导的HL-60/ADM细胞凋亡。结论：1.HL-60/ADM耐药细胞株中线粒体ATPsyn-β表达显著低于其亲本的HL-60细胞。2.下调线粒体ATPsyn-β使HL-60细胞对阿霉素的耐药性增加,并阻滞细胞凋亡；上调ATPsyn-β基因表达可以逆转HL-60/ADM细胞对阿霉素的耐药,促进耐药细胞的凋亡。3.ATPsyn-β可影响HL-60及其耐药细胞株MRP基因的表达。第四章急性髓系白血病患者ATPsyn-β表达高低与预后分析目的：总结118例急性髓系白血病患者实验室指标与预后风险。分析AML患者ATPsyn-β表达与疗效的关系。方法：利用Kaplan-Meier生存分析、t检验、Wilcoxon符号秩和检验等对年龄、ECOG评分、白细胞计数与CR期、OS的关系进行分析。分析ATPsyn-β表达与预后的关系。结果：1、118例AML患者,45岁以下组的平均缓解期和总生存期分别为16.06个月和25.17个月,45岁以上组分别为9.49个月、16.98个月,差异有统计学意义。ECOG评分0-2级的患者生存状况要好于3-4级者,P<0.01。白细胞计数与OS呈显著负相关。以30×109/L为切点,白细胞计数增高组预后较差,两组的平均OS分别12.25个月和25.65个月,P<0.05。2、不同性别、年龄及体能状态的患者ATPsyn-β的表达差异不显著,ATPsyn-β的表达量与FAB分型关系不明显。初诊AML患者ATPsyn-β低表达组的中位缓解期为4个月,而ATPsyn-β高表达组的中位缓解期为8个月,P<0.05；截止末次随访日期,两组在总生存期上无明显差异。复发难治患者ATPsyn-β的表达高低与总生存期长短显著相关,复发难治AML患者ATPsyn-β mRNA低表达者总生存期较短,为10.68±7.87个月,而高表达者为21.28±1.26个月,P<0.05。结论：1、年龄、体能状态、初诊时白细胞计数高低是急性髓系白血病患者预后的主要危险因素。2、线粒体ATPsyn-β是一个新的提示急性髓系白血病预后的指标,ATPsyn-β表达水平的高低与患者缓解期的长短呈显著相关。复发难治AML患者ATPsyn-β mRNA低表达者总生存期较短。更多还原

【Abstract】 BackgroundLeukemia is a group of heterogeneous diseases characterized by either a block in differentiation, aberrant and accelerated growth, or a combination of both with considerable diversity in terms of clinical behavior and prognosis. Acute myelogenous leukemia (AML) is the most common type of leukemia in adults. The main therapy is still chemotherapy induction and consolidation. However, nearly70%of responding patients will eventually relapse and die of the disease. One of the major causes of failure in the treatment is drug resistance. But the mechanisms underlying the development of multidrug resistance in AML are not fully understood. Therefore, it is significant to explore novel molecular targets for drug-resistant reversal in AML patients.Recently, a rebirth of the interest in the energetic metabolism of cancer spurred, and mitochondria has become a central player. The mitochondrial ATP synthase is the enzyme complex that carries out the synthesis of ATP. The β subunit of mitochondrial ATP synthase (ATPsyn-β) is the rate-limiting component of mitochondria oxidative phosphorylation. Down-regulated expression of mitochondrial ATPsyn-β has been reported in many types of tumors,including colon、lung、breast、 esophagea、stomach and kidney.Our previous study shows that down-regulated expression of mitochondria ATPsyn-β is associated with the development of drug resistance in chronic myeloid leukemia (CML). Here, we studied the expression of mitochondrial ATPsyn-β and the activity of mitochondrial ATPase in AML patients.Chapter Ⅰ The expression of mitochondrial ATPsyn-β on bone marrow mononuclear cells and CD34+cells form AML patients and the correlation between ATPsyn-β level and sensitivity to adriamycin in vitro.Objective:To investigate the expression of mitochondrial ATPsyn-β in AML patients and the association with chemotherapy response.Methods:1. Bone marrow samples were collected from118non M3AML patients (42of relapsed/refractory,38of complete remission duration,38of newly diagnosis) and20patients with benign hematological diseases(for control)from Oct,2010to Jun,2012. Expression of mitochondrial ATPsyn-β mRNA and protein in bone marrow mononuclear cells (BMMCs) of AML patients were detected by RT-PCR, western blot and flow cytometry respectively.2. The purified CD34positive cells were acquired from BMMCs of20AML patients using the CD34positive selection immunomagnetic beads. Then the expression of ATPsyn-β mRNA and protein were analyzed by RT-PCR and western blot.3. Sensitivity of BMMCs to adriamycin in relapsed/refractory and remission duration AML patients was detected by MTT assay in vitro. Correlation between expression of ATPsyn-β and adriamycin sensitivity in the two groups was used linear correlation to analysis.Results:1. Expression of ATPsyn-β mRNA and protein in AML patients (n=118) was downregulated (P<0.01). 2. Different levels of mitochondrial ATPsyn-β mRNA and protein were detected in AML patients. Compared with remission duration patients(n=38), expression of ATPsyn-β in relapsed or refractory AML patients(n=42)was significantly downregulated both in BMMCs and CD34positive cells, P<0.05.3. The relative level of ATPsyn-β mRNA was higher in CD34+cells than in CD34-cells, P<0.05.4. IC50value of adriamycin to BMMCs from relapsed/refractory and remission duration AML patients was13.69μM and1.549μM respectively.5. The mRNA and protein expression of ATPsyn-β significantly inversely correlated with IC50of adriamycin to BMMCs in relapsed/refractory AML patients, P<0.05.Conclusion:The mRNA and protein expression of mitochondrial ATPsyn-β was significantly downregulated in non-M3AML patients. Compared with complete remission patients, the mRNA and protein level of ATPsyn-β in relapsed/refractory AML patients were lower in both bone marrow mononuclear cells and CD34positive cells. And this decrease correlated with resistance of primary leukemia cells to adriamycin in vitro, suggesting that down-regulation of ATPsyn-β in AML is associated with drug resistance. Chapter II The activity of mitochondrial ATP synthase in drug-resistant leukemia cell lines and primary cells of AML patientsObjective:To investigate the activity of mitochondrial ATP synthase in AML patients and the clinic significance. Methods:After the isolation of mitochondia, the activity of ATPase in adriamycin resistant cell lines(K562/A02、HL-60/ADM) and BMMCs of AML patients was measured in the direction of ATP hydrolysis using a kit.Results:1. The mitochondrial ATPase activity of K562/A02(36.70±7.47U) and HL-60/ADM cells(38.22±2.00U) was lower than K562(65.43±1.31U) and HL-60cells(132.06±14.65U), P<0.05.2. The mitochondrial ATPase activity in relapsed or refractory samples (n=26) was obviously reduced when compared with those of complete remission samples (n=16). The mean value was20.33U vs.31.17U, P<0.05.3. Mitochondrial ATPsyn-β mRNA expression showed a positive correlation with ATPase activity (rs=0.294, P=0.031).Conclusion:The activity of mitochondrial ATP synthase was down-regulated in adriamycin resistant cell lines and relapsed/refractory AML patients. Mitochondrial ATPsyn-β mRNA expression had a positive correlation with ATPase activity. Chapter Ⅲ Expression of mitochondrial ATPsyn-β in adriamycin resistance AML cell lines and its effect on growth inhibition and apoptosis inductionObjective:To explore the influence of ATPsyn-β on proliferation-inhibiting capability and apoptosis-inducing effect of adriamycin in leukemia cells. To analyse whether the change of ATPsyn-β level could lead to or reverse drug resistance. Methods:1. Expression of mitochondrial ATPsyn-β in HL-60and HL-60/ADM cell lines was detected by RT-PCR, western blot and flow cytometry.2. Afer inhibition of ATPsyn-β expression by RNA interference or increase it by gene transfection, HL-60and HL-60/ADM cells were treated with different concentrations of adriamycin, then (1)cell viability was analyzed with MTT colorimetric assay test;(2) cellular uptake of adriamycin was detected by flow cytometry;(3) morphologic character of apoptosis was evaluated by Wrights-Giemsa staining and Hoechst33258staining;(4)the percentage of earlier apoptosis cell was analyzed by flow cytometry;(5)expression of MRP mRNA relative level was detected by RT-PCR.Results:1.The expression of mitochondrial ATPsyn-P in HL-60/ADM cells was lower than HL-60cells, and MRP mRNA expression was higher.2.Overexpression of ATPsyn-β in HL-60/ADM cell line restored cells’ sensitivity to adriamycin, promoted earlier cell apoptosis and decreased the expression levels of MRP. In contrast, suppression of ATPsyn-β expression weakened cell’sensitivity to adriamycin, blocked cell apoptosis and increased the expression of MRP.Conclusion:The expression of mitochondrial ATPsyn-p in HL-60/ADM cells was downregulated. Inhibition of mitochondrial ATP synthase β subunit led to increased chemo-resistance and apoptotic resistance in HL-60cells and up regulation of mitochondrial ATPsyn-β restored chemo-sensitivity and promoted earlier cell apoptosis in HL-60/ADM cells. Chapter Ⅳ Expression of ATPsyn-P in AML patients with prognostic analysisObjective:To analyze the impact of established prognostic factors on the clinical outcome in patients with AML. To determine the prognostic value of ATPsyn-β in AML patients.Methods:We analyzed3variables for their impact on complete remission (CR) duration and overall survival (OS) in118AML patients, including age, performance status and WBC counts. We also determined the role of ATPsyn-P as a poor prognostic factor in AML patients.Results:1. Multivariate analysis determined old age (older than45years), poor PS (score3-4), leukocytosis (WBC>30×109) as poor prognostic factors for OS.2. Much of the relation between ATPsyn-β and therapeutic resistance was observed. The median CR duration in newly diagnosis AML patients with low ATPsyn-β mRNA expression was4months, shorter than that in high ATPsyn-β expression patients (eight months). The overall survival of relapsed/refractoty AML patients with low ATPsyn-β mRNA expression was10.68±7.87months, shorter than that with high lower ATPsyn-P mRNA expression(21.28±1.26months), P<0.05。Conclusion:1. Age, performance status, WBC counts onset were independent prognostic factors for clinical outcome in AML.2. As a prognostic factor for survival, low level of mitochondrial ATPsyn-P was associated with an increased relapse risk, shorter CR duration and overall survival.更多还原