【作者】 支蕾；

【导师】 庞天翔； 王建祥； 秘营昌； 王敏； 饶青；

【作者基本信息】 中国协和医科大学 ， 病理学与病理生理学， 2010， 博士

【摘要】 研究目的：白血病干细胞(LSCs)是白血病发生和耐药、复发的重要原因,其具有自我更新能力。目前的研究表明LSCs存在于CD34+/CD38-/CD123+细胞群中,但LSCs的精确表面标记尚不清楚。LSCs通过粘附分子与骨髓基质相互作用可能会促进白血病细胞的自我更新失调和凋亡受阻,这可能是LSCs能够逃逸化疗的机制之一。因此研究骨髓微环境(niche)相关的细胞表面标记,有助于靶向LSCs治疗靶点的发现。本实验中我们研究粘附分子N-cadherin,Tie2和CD44在白血病干细胞中的表达及化疗对上述抗原表达富集的影响,探讨其是否为LSCs的潜在标志。研究方法：用流式细胞术检测63例急性髓系白血病(acute myeloid leukemia, AML)患者骨髓单个核细胞(BMMNCs)化疗前后N-cadherin、Tie2、CD44、CD34、CD38和CD123的表达,观察LSCs (CD34+/CD38-/CD123+细胞群)中N-cadherin, Tie2和CD44的表达情况。联合免疫表型和荧光原位杂交技术,对4例伴有t(8；21)移位患者的BMMNCs,分选纯化CD34+/CD38-/CD123+/N-cadherin+和CD34+/CD38-/CD123+/Tie2+细胞群和CD34+/CD38-/CD 123+/N-cadherin-和CD34+/CD38-/CD 123+/Tie2-细胞群,检测AML1/ETO的表达情况。实时定量PCR技术,对5例AML患者的BMMNCs,分选纯化CD34+/CD38-/CD123+/N-cadherin+和CD34+/CD38-/CD123+/Tie2+细胞群和CD34+/CD38-/CD123+/N-cadherin-和CD34+/CD38-/CD123+/Tie2-细胞群,检测耐药基因MDR1mRNA的表达水平情况。结果：①在AML患者BMMNCs中N-cadherin和Tie2表达阳性的LSCs比例在停化疗当天与化疗前比较无明显差异,在停化疗后第7天分别由0.17%、0.02%升高到0.6%、0.94%(P<0.05,P<0.01),而CD44阳性细胞的LSCs比例由17.28%降低到2.25%(P<0.05)(中位数)；②在LSCs中,N-cadherin,Tie2和CD44表达阳性细胞比例在停化疗的第7天较化疗前分别升高了38.17(P=0.03)、210(P<0.01)、1.01(P=0.15)倍(中位数),提示N-cadherin+和Tie2+的LSCs细胞能抵抗化疗,可能是潜在的LSCs标志；③共表达N-cadherin和Tie2的LSC细胞群化疗后富集比例高于共表达CD44的LSCs细胞群,提示共表达CD44的LSCs细胞群可能比较成熟,而共表达N-cadherin和Tie2的LSCs细胞群可能是真正的LSC细胞群；④未缓解病例组中,共表达N-cadherin、Tie2和CD44的LSCs细胞群在治疗前和停化疗后第21天的比例明显高于缓解组病例,提示共表达N-cadherin、Tie2和CD44的LSCs细胞群可能是微量残留细胞,与预后不良有关；⑤细胞遗传学分组、CD56表达强弱分组及治疗前外周血白细胞计数分组显示共表达N-cadherin和Tie2的LSCs细胞群在预后不良组中的比例明显高于良好组,进一步提示N-cadherin+和Tie2+的LSCs与预后不良有关；⑥N-cadherin和Tie2在LSCs细胞群中的表达水平与临床骨髓涂片的幼稚细胞比例显著相关,提示检测共表达N-cadherin和Tie2的LSCs细胞群可作为临床评估预后及微量残留病的指标；⑦分选纯化的CD34+/CD38-/CD123+/N-cadherin+和CD34+/CD38-/CD123+/Tie2+细胞群,检测到AML1/ETO的融合信号分别是87.53%和92.18%,而分选纯化的CD34+/CD38-/CD123+/N-cadherin-和CD34+/CD38-/CD123+/Tie2-细胞群,检测到AML1/ETO的融合信号分别是72.36%和80.87%,提示AML1/ETO表达在干细胞水平,其恶性程度更高。⑧在分选纯化的CD34+/CD38-/CD123+/N-cadherin+细胞中检测到N-Cadherin mRNA的表达,而在CD34+/CD38-/CD123+/N-cadherin-细胞群中未检测到N-Cadherin mRNA的表达；在分选纯化的CD34+/CD38-/CD 123+/Tie2+和CD34+/CD38-/CD123+/Tie2-细胞群中,一位AML患者的标本检测到N-Cadherin mRNA表达,而另两位AML患者标本中未检测到N-Cadherin mRNA表达,提示部分患者LSCs中共表达N-Cadherin和Tie2；⑨分选纯化的CD34+/CD38-/CD123+/N-cadherin+和CD34+/CD38-/CD123+/Tie2+细胞群,检测到MDR1 mRNA,表达水平分别是8.16±1.07和4.18±3.93,而分选纯化的CD34+/CD38-/CD123+/N-cadherin-和CD34+/CD38-/CD123+/Tie2-细胞群,检测到MDR1mRNA,表达水平分别是0.06±0.06和0.46±0.49,进一步说明N-cadherin+和Tie2+的CD34+/CD38-/CD123+比N-cadherin-和Tie2-的CD34+/CD38-/CD123+对化疗更不敏感。结论：共表达粘附分子N-cadherin和Tie2的LSCs细胞群具有抵抗化疗的作用,能够通过化疗而富集,并表达AML1/ETO恶性分子和耐药基因MDR1,可以作为识别LSCs的潜在标志。研究目的：富含鸟嘌呤的寡核苷酸(G-rich oligonucletides, GRO)通过非反义途径,具有抑制肿瘤及白血病细胞生长和增殖的功能,诱导肿瘤细胞停滞于细胞周期的S期。GRO可以形成独特的G—四聚体的稳定折叠结构,可以与特异性核蛋白——核仁素结合。核仁素蛋白的表达水平反映细胞增殖状态,增殖旺盛的肿瘤细胞中核仁素蛋白表达水平高。有研究发现,细胞受到外来打击后,核仁素能与P53结合形成复合物。为研究GRO抗白血病细胞增殖的作用及机制,本实验探讨了GRO、核仁素和P53的相互作用,尤其是P53基因及其相关信号途径是否影响GRO的生物学作用。研究方法：设计GRO和其对照药物富含胞嘧啶的寡核苷酸(C-rich oligonucletides, CRO)。通过慢病毒载体将P53基因转入U937细胞系(缺失表达P53),分别用PBS、GRO及CRO处理U937细胞系、转染空载体和转染P53的U937细胞,从抑制增殖、促进细胞凋亡、诱导细胞周期停滞及对核仁素蛋白和周期蛋白的作用等方面检测P53基因对GRO药物作用的影响。研究结果：(1)GRO具有明显抑制细胞增殖,促进凋亡及诱导细胞周期停滞于S期的功能。(2)激光共聚焦结果显示：U937细胞中,GRO能与核仁素蛋白结合,可能是GRO发挥作用的机制。转染P53的U937细胞中, GRO的摄入减少。(3)转染P53后削弱GRO抑制细胞增殖、促进凋亡及诱导细胞周期停滞的作用。(4)GRO处理U937细胞24h后,CDK2蛋白表达水平增加,48h后CDK2蛋白表达水平降低,而GRO处理转染P53的U937细胞,细胞周期蛋白的表达出现滞后现象。研究结论：GRO可以抑制白血病细胞增殖、促进细胞凋亡及诱导细胞周期停滞于S期,而转染P53后,GRO摄入减少,其抑制增殖、促进细胞凋亡、诱导细胞周期停滞的生物学作用降低；GRO诱导细胞周期停滞的机制可能与CDK2有关,24h时CDK2表达增加,促进细胞进入S期,48h后CDK2表达降低使细胞停滞于S期。而P53的表达影响了细胞周期,细胞周期蛋白的表达出现滞后现象。本实验提示,P53与核仁素结合,影响了GRO的生物学作用,解释了GRO、P53、核仁素三者的相互关系,并对GRO的临床使用具有指导意义。更多还原

【Abstract】 Objective:Leukemia stem cell (LSC) is the main cause of leukemogenesis, drug-resistance and relapse of leukemia. They have the potential of self-renewal. It has been recognized that LSCs reside within the CD34+/CD38-/CD123+ compartment. However, the pure population of LSCs in patients with AML has not been identified. The interaction mediated by adhesion molecules between LSCs and BM stromal cells played a role in the disturbed self-renewing and inhibited apoptosis of LSCs, which may be one of the mechanism of escape from chemotherapy. Therefore, to study the cell surface makers related to niche on LSCs is helpful for the potential target therapy in the future. In this study, expression of the three adhesion molecules, the N-cadherin, Tie2 and CD44 in the proportion of CD34+/CD38-/CD123+ LSCs from AML patients before and after chemotherapy were analyzed. Whether the N-cadherin, Tie2 and CD44 could be the potential markers for identification of LSCs were investigated.Methods:Bone marrow mononuclear cells (BMMNCs) from the 63 AML patients were obtained at different time points including the day before chemotherapy (at time of diagnosis, Pre), one day after the induction chemotherapy (Post-Day1), seven days after the induction chemotherapy (Post-Day7) and twenty one days after the induction chemotherapy (Post-Day 21), and analyzed by fluorescence activated cell sorting (FACS) for the expression of N-cadherin, Tie2, CD44, CD34, CD38 and CD123. And the proportions of N-cadherin, Tie2 and CD44 in LSCs (CD34+/CD38-/CD123+ cells compartment) were investigated. Combination with immunophenotyping and fluorescence in situ hybridization (FISH) analysis, N-cadherin and Tie2 positive CD34+/CD38-/CD123+ LSCs proportions of BM samples derived from 4 AML-M2 patients were isolated by FACS sorting for expression of AML1/ETO. N-cadherin and Tie2 positive CD34+/CD38-/CD123+ LSCs proportions of 5 AML patients were isolated by FACS sorting for expression of MDR1 mRNA by real-time PCR.Results:①In BMMNCs of AML patients, no obvious increase in the proportions of LSCs is occurred at Post-Day 1. With the subsequent effect of chemotherapy and the complete elimination of chemosensitive cells, the proportions of N-cadherin and Tie2 positive CD34+/CD38-/CD123+ LSCs increased from 0.17% and 0.02% to 0.6% and 0.94% at Post-Day 7, respectively (P<0.05, P<0.01), but the proportions of CD44 positive CD34+/CD38-/CD123+ LSCs decreased from 17.28% to 2.25%(P<0.05) (median);②In CD34+/CD38-/CD123+ LSCs, the fold of increase in the proportions of N-Cadherin, Tie2 and CD44 positive in CD34+/CD38-/CD123+ LSCs populations at Post-Day 7 could be clearly demonstrated as 38.17 (P=0.01),210 (P=0.03) and 1.01 (P=0.15), respectively. Results further illustrated that CD34+/CD38-/CD123+/N-Cadherin+ and CD34+/CD38-/CD123+/Tie2+ cells compartments were more resistant to chemotherapy than CD34+/CD38-/CD123+/CD44+ cells compartment, and the former two cells compartments have the properties of LSCs, they may be the true LSCs;③The enrichment capability of N-cadherin and Tie2 positive LSCs compartment were stronger than CD44 positive LSCs compartment, which indicated N-cadherin and Tie2 positive LSCs compartment may be the true LSCs;④In patients did not achieve CR (non-remission, NR) group, the proportions of CD34+/CD38-/CD123+/N-Cadherin+, CD34+/CD38-/CD123+/Tie2+ and CD34+/CD38-/CD123+/CD44+ cells compartments in BMMNCs showed a tendency to be higher than that in complete remission (CR) group at the time of diagnosis (P>0.05), which proposed that N-Cadherin, Tie2 and CD44 expression may serve as markers for MRD cells that may allow their identification for early detection of relapse;⑤At diagnosis, the proportions of N-Cadherin and Tie2 positive CD34+/CD38-/CD123+cells compartment were higher in cytogenetic unfavorable group, the white blood cell (WBC) count of more than 30×109/L and CD56 positive expression, which further proposed that N-Cadherin, Tie2 and CD44 expression may be associated with poor prognosis;⑥The proportions of N-Cadherin, Tie2 and CD44 positive CD34+/CD38-/CD123+ cells compartment were correlated with BM blast percentages at Post-Day 7, which proposed that N-Cadherin, Tie2 and CD44 co-expression may serve as markers for MRD cells that may allow their identification for early detection of relapse;⑦(87.75±6.01)%and (72.36±16.47)%AML1/ETO fusion signals were detected in the CD34+/CD38-/CD123+/N-cadherin+and CD34+/CD38-/CD123+/N-cadherin- LSCs populations, respectively. And (92.18±8.23)%and (80.87±16.45)%AML1/ETO fusion signals were detected in the CD34+/CD38-/CD123+/Tie2+ and CD34+/CD38-/CD123+/Tie2- LSCs populations, respectively, which suggesting that they might present at LSCs populations.⑧N-cadherin mRNA expression were detected in CD34+/CD38-/CD123+/N-Cadherin+ cells counterparts, and none in CD34+/CD38-/CD123+/N-Cadherin- cells counterparts, which would be as positive control. However, in 3 samples of Tie2 LSCs counterparts, N-cadherin mRNA expression was detected in both Tie2+ and Tie2- LSCs counterparts in 1 sample, but none in Tie2+ and Tie2- LSCs counterparts of another 2 samples. The results indicated that the expression of Tie2 and N-cadherin seems overlapped in some patients.⑨The relative quantity of MDR1 expression in N-cadherin+ and Tie2+ LSCs counterparts were higher than that of N-cadherin- and Tie2- LSCs counterparts(8.16±1.07 and 4.18±3.93 vs 0.06±0.06 and 0.46±0.49, respectively). These results could further confirm the finding that N-cadherin+ and Tie2+ LSCs counterparts are more resistant to chemotherapy than their negative LSCs counterparts.Conclusions:N-cadherin+ and Tie2+ expressed CD34+/CD38-/CD123+ LSCs populations could be less sensitive to chemotherapy and be enriched by chemoresistance. However, AML1/ETO and MDR1 were found in the N-cadherin+ and Tie2+ positive LSCs. It is suggested that N-Cadherin and Tie2 may be the potential markers of LSCs and also be the candidate therapeutic targets. Objective:G-rich oligonucleotides (GROs) have been demonstrated to inhibit proliferation and induce cell cycle arrest at S phase in tumor cell lines. The biological activity of GROs results from their binding to specific cellular proteins, nucleolin. Increases in nucleolin levels in unstressed cells led to activation of P53. It has been previously identified that activation of P53 could stimulate nucleolin-P53 complex formation under stress. GROs showed anti-proliferation activity through a non-antisense way, due to their stable G-quartets structure formation. This stable structure was found to interact with a specific cellular protein, nucleolin. Increases in nucleolin levels in unstressed cells led to activation of P53. And the biological activity of GROs results from their binding to nucleolin, as well as, nucleolin can interact with P53. The relationship among the effect of GRO, nucleolin and P53 is unknown yet. To investigate its mechanism of GRO, the effects of p53 on the biological function of GROs were studied.Methods:GROs and C-rich oligonucleotides (CROs) as control were performed to determine the function of GRO in leukemia cell lines. Western blot analysis was employed to identify the P53 protein expression in U937 cell lines, which have no P53 protein expression. HIV-based lentivector expression vector pCDH1-MCS1-EF1-copGFP containing full-length coding sequence (CDS) region of p53 was constructed and transfected to express p53 in U937 cell lines. Apoptosis assay by flow cytometry, cell cycle and MTT were performed to determine the function of GRO in U937 cell lines induced by the apoptotic stimuli, P53.Results:1) GRO was shown to have significant anti-proliferative activities, inducing apoptosis and cell cycle arrest at S phase in U937 cells (human monocytic leukemia cells) which have no P53 protein expression.2) By the laser scanning confocal microscope (LSCM) assay, we verify for the first time the co-localization between GRO and nucleolin inside U937 cells. However, P53 expression could cause the partial formation of nucleolin-P53 complexion by exposure to GRO.3) The effect of GRO was weakened in U937 cells with P53 expression.4) In U937 cells with GRO treatment, increased Cdk2 led the cells enter S phase. Then, Cdk2 began to decease that made the cells arrest in S phase. Expression of P53 in U937 cells affects the alteration of cell cycle and related regulation proteins. Conclusion:GRO was shown to have significant anti-proliferative activities, inducing apoptosis and cell cycle arrest at S phase in U937 cells. And in U937 cells with GRO treatment, increased Cdk2 led the cells enter S phase. Then, Cdk2 began to decease that resulted in the cells arrest in S phase. However, the effect of GRO was weakened in U937 cells with P53 expression, which may be related with the decreased intake of GRO because of the partial formation of nucleolin-P53 complexion.更多还原