【作者】 杨晶；

【导师】 黄士昂；

【作者基本信息】 华中科技大学 ， 内科学， 2007， 博士

【摘要】 第一部分利用定量流式细胞术比较PTEN在急性白血病患者和正常人中的表达差异目的:研究肿瘤抑制基因PTEN在急性白血病患者和正常人骨髓细胞中的表达情况,并比较其差异。方法:收集36例初发急性白血病患者和5例正常人的骨髓细胞,利用定量流式细胞术,通过Kolmogorov-Smirnov (KS)分析,检测PTEN表达情况。结果:正常人骨髓淋巴细胞、粒细胞、单核细胞PTEN表达的D值分别为(0.93±0.05),(0.97±0.02)和(0.95±0.03)。36例初发的急性白血病患者中,26例急性髓细胞白血病(AML)患者(1例M0,2例M1,11例M2,3例M3,6例M4,2例M5,1例M6)的骨髓原始细胞均检测到PTEN的表达,除1例M4和2例M5患者PTEN表达与正常对照组的接近,其余患者PTEN表达均较正常对照组降低,但是各个样本PTEN的表达表现出很大的差异性。8例急性B淋巴细胞白血病(B-ALL)患者的骨髓原始细胞PTEN的表达明显缺失,D值为(0.51±0.05)。2例急性T淋巴细胞白血病(T-ALL)患者的骨髓原始细胞PTEN表达的D值为( 0.74±0.02)。结论:1.AML患者骨髓原始细胞均检测到PTEN的表达,但较正常对照组下调,且各个样本PTEN的表达表现出很大的差异性。AML患者骨髓原始细胞PTEN表达和FAB分型之间的联系是否具有统计学意义还需进一步研究。2.PTEN的表达明显下调或缺失在B-ALL中是一个常见现象。第二部分B-ALL患者PTEN表达缺失及其启动子甲基化状态关系的研究目的:探讨抑癌基因PTEN在急性B淋巴细胞白血病(B-ALL)患者外周血单个核细胞中的表达缺失及其与启动子甲基化状态之间的关系。方法: 24例初发B-ALL患者的外周血单个核细胞和25例正常人外周血单个核细胞,利用定量流式细胞术,通过Kolmogorov-Smirnov (KS)分析,检测PTEN表达情况后,提取基因组DNA,采用MSP方法检测PTEN启动子甲基化状态。进而将外周血单个核细胞在体外培养过程中,以DNA甲基化转移酶抑制剂5-氮-2’脱氧胞嘧啶进行处理,观察其对B-ALL标本中PTEN的表达的影响。结果:25例健康供者外周血单个核细胞均表达PTEN,KS分析时D值为0.941±0.017,而24例B-ALL患者中22例(91.6%)外周血单个核细胞PTEN的表达明显缺失,D值仅为0.546±0.115,两者差异具有显著性意义(P<0.01);正常人和PTEN高表达的B-ALL患者外周血单个核细胞中未发现PTEN启动子发生甲基化修饰,而PTEN表达缺失的B-ALL患者中有5例(22.7%)出现PTEN启动子甲基化,将其单个核细胞培养时以5-氮-2’脱氧胞嘧啶处理4天,PTEN表达得以明显升高。讨论:1.PTEN的表达明显下调或缺失在B-ALL中很常见2.PTEN启动子甲基化导致的PTEN表达缺失对B-ALL的发生可能起到重要的作用。第三部分STI571治疗2例携带e19a2的加速期CML患者的细胞遗传学改变目的:研究携带e19a2融合转录方式CML患者的临床特征及其与临床预后之间的关系,以及STI571对其治疗的效果。方法:两例加速期CML患者行STI571治疗,检测e19a2的表达情况,并定期进行骨髓细胞遗传学、巢式RT-PCR、FISH检测。结果:两例患者均携带e19a2,第1例患者经9个月的STI571治疗(600mg/天)后,细胞遗传学检查未见Ph染色体,巢式RT-PCR也仅检测到极少量的e19a2融合基因。第2例患者经6个月的STI571治疗(600mg/天)后,细胞遗传学检查未见Ph染色体和+der(22)t(9;22),通过FISH仅在5%的间期核中检测到+der(22)t(9;22)。讨论:1.e19a2可能与临床预后不良的CML相关联。2.STI571治疗对加速期的携带e19a2的患者依然有效。更多还原

【Abstract】 PARTⅠA Comparison of PTEN Expression Difference on Both Normal Person and Acute Leukemia Patients by Quantitative Flow Cytometry AnalysisObjective To study and comparison of a novel tumor suppressor gene PTEN expression difference on bone marrow cells from both healthy person and acute leukemia patients Methods Bone marrow cell samples were Collected form both 5 normal donors and 36 primary acute leukemia patients, Used Kolmogorov– Smirnov (KS) statistical test to characterize the expression of PTEN in quantitative flow cytometry analysis.Results Lymphocytes, granulocytes and monocytes of five normal bone marrow samples showed comparable levels of PTEN expression, with average D-values of 0.93±0.05, 0.97±0.02, and 0.95±0.03, respectively. By the test of Bone marrow blast cell simples from 36 primary acute leukemia patients, PTEN expression could be detected In the 26 patients with de novo AML (1 M0, 2 M1, 11 M2, 3 M3, 6 M4, 2 M5 and 1 M6) simples tested. Except 1 M4 and 2 M5 patents samples showed approximate PTEN expression level compared with Normal samples, PTEN expression level of other Patient samples significantly lowers than that in normal cell populations. Whereas, particularly PTEN expression difference was detected in samples tested. 8 B-ALL patient samples showed spectacularly low down PTEN expression level with D-values of 0.51±0.05. compared with normal samples. 2 T-ALL patients samples with D-values of 0.74±0 02.Conclusion 1. All AML patients showed perceptible PTEN expression whereas particularly low level than normal samples, differences observed in samples respectively. This observation needs to be validated in more patients, and its implications should be further investigated whether PTEN expression level correlated with leukemia FAB subtype.2. absent/low PTEN expression is a frequent event in B-ALL.PARTⅡThe Investigation of PTEN Expression and Its Promoter Methylation in B-ALL PatientsObjective To study the expression of tumor suppressor PTEN in peripheral blood mononuclear cells of B-ALL patients and involvement of promoter methylation in the loss of PTEN expression in these patients.Methods Kolmogorov– Smirnov (KS) statistical test was used to characterize the expression of PTEN in peripheral blood mononuclear cells from both healthy donors and B-ALL patients in quantitative flow cytometry analysis. PTEN Promoter Methylation status of genomic DNA extracted from the peripheral blood mononuclear cells was determinded by MSP analysis.The peripheral blood mononuclear cells were treated with 5-Aza-2’deoxycytidine for 4 days to study its effect on restoring PTEN expression in B-ALL peripheral blood mononuclear cells.Resuits Compared with the high level PTEN expression in healthy donors(D value 0.941±0.017), PTEN expression could barely be detected in 22(91.6%) B-ALL patients ( D value 0.546±0.115). PTEN Promoter Methylation was detected in 5 B-ALL patients(22.7%)but not in any healthy donor. Moreover, 5-Aza-2’deoxycytidine treatment could restore the PTEN epxression in these patients.Conclusion 1.Low/absent expression of PTEN is a frequent event in B-ALL patients2.promoter Methylation should contribute to the loss of PTEN epression in some of B-ALL patients. PartⅢThe Investigation of Early Cytogenetic Response to Imatinib in Two Patients with CML at Accelerated Phase and Carrying the e19a2 BCR-ABL TranscriptObjective To study implications of e19a2 and CML clinic prognosis, and Imatinib’s therapeutic effect on CML patients with e19a2 transcriptsMethod 2 accelerated phase CML patients had been treated by STI571 and tested with e19a2 expression. Periodic Bone Marrow Cytogenetic Analysis, Nested Reverse Transcription Polymerase Chain Reaction Analysis and FISH Analysis also included. Results e19a2 expression detected in 2 patient samples at first begin. No Ph chromosome detected in sample of No.1 Patient by cytogenetic analysis after 9 months STI571 treatment (600mg/Day), only very small quantity of e19a2 fused gene was detected in sample. The other patient was treated by STI571 for 6 months (600mg/Day), no Ph chromosome and +der(22)t(9;22) detected by cytogenetic analysis, +der(22)t(9;22) was detected in only 5% Interphase nuclei.Conclusion 1.the presence of e19a2 transcript might be associated with a unfavorable prognosis in CML.2.imatinib treatment could be also valuable for CML patients with e19a2 rearrangements, even in accelerated phase.更多还原