【作者】 刘娟；

【导师】 赵谢兰；

【作者基本信息】 中南大学 ， 内科学， 2012， 硕士

【摘要】 背景与目的：慢性粒细胞性白血病是骨髓造血干细胞克隆性增殖形成的恶性肿瘤,占成人白血病的15%-20%。伊马替尼是一种BCR/ABL融合基因酪氨酸激酶的竞争性抑制剂,在治疗慢性粒细胞性白血病取得显著的效果,目前NCCN(?)旨南已明确伊马替尼是CML的一线治疗药物。虽然伊马替尼治疗CML取得良好的效果,但部分患者在治疗初期或是治疗过程中出现伊马替尼耐药。伊马替尼可能的耐药机制有多种,目前认为主要的耐药机制是BCR/ABL基因扩增及过度表达和BCR/ABL基因突变。然而有多个对伊马替尼耐药的K562细胞株的研究发现：在这些伊马替尼耐药的K562细胞株中未出现BCR/ABL基因扩增及过度表达和(?)BCR/ABL基因突变,却发现存在SFKs家族激酶中的Lyn高表达。因此,本研究对76例慢性粒细胞性白血病患者骨髓单个核细胞中的Lyn表达水平进行监测,分析其与临床特征、临床疗效、染色体异常的关系,来探讨Lyn在伊马替尼耐药的CML中的作用。方法：采用Western bloting测定76例慢性粒细胞性白血病和10例正常人造血干细胞的骨髓单个核细胞中Lyn的表达,分析其与临床特征、伊马替尼耐药的关系。采用RT-PCR对慢性粒细胞性白血病患者BCR/ABL融合基因进行检测,分析其与Lyn的关系。采用R显带计数对其中10例慢粒耐伊马替尼患者进行核型检测,分析其与Lyn表达的关系。采用SPSS17.0统计软件对数据进行t检验(或非参数检验)、Speannan秩相关(或Pearson相关),P<0.05表明差异有统计学意义。结果：1、Lyn与伊马替尼耐药的关系伊马替尼耐药组Lyn表达明显高于正常对照组、初治组及伊马替尼治疗有效组(P=0.0025、P=0.0014、P=0.0001),初治组、伊马替尼治疗有效组与正常对照组无明显差别(P=0.310、P=0.2355)。2、BCR/ABL基因的定量分析研究检测了初治组、有效组、耐药组BCR/ABL表达情况,结果发现初治组和耐药组BCR/ABL表达明显高于有效组(P=0.035、P=0.026)。3、Lyn与BCR/ABL表达的关系对Lyn和BCR/ABL表达进行相关性分析,发现两者无明显相关性(P=0.278)。4、Lyn与临床特征76例CML均表达Lyn。Lyn表达与患者性别(P=0.068)、年龄(P=0.070)、平均血红蛋白水平(P=0.869)、平均血小板计数(P=0.968)、外周血幼稚细胞比例(P=0.873)、脾脏大小(P=0.647)无明显相关。CML患者初诊时白细胞计数>100×109/L者Lyn表达明显增高(P=0.0465),差异有统计学意义。5、Lyn与伊马替尼耐药的CML染色体的关系对10例伊马替尼耐药的CML进行染色体检查,发现1例患者存在t(6；22)和t(2；9)改变,与Ph染色体共存,其余9例患者仅存在Ph染色体改变。将CML患者按染色体改变分为两组(Ph染色体组和Ph染色体外的其他克隆性染色体异常组),两组患者间Lyn表达无明显差异(P=0.487)。结论：1、CML患者和正常人均表达Lyn。2、Lyn在伊马替尼耐药的CML中表达明显增高。3、Lyn表达与患者年龄、性别、平均血红蛋白水平、平均血小板计数、外周血幼稚细胞比例、脾脏大小无明显相关。Lyn表达与CML患者初诊时白细胞计数明显增高有关。4、Lyn表达与BCR/ABL无明显相关。5、Lyn表达与染色体核型无明显相关。更多还原

【Abstract】 Background and Objective:Chronic myelogenous leakemia (CML) is a malignant tumor which is composed of clonal preliferation of hematopoietic stem cell and is found in15%-20%of patients with leakemia.Imatinib is a protein tyrosine kinase competitive inhibitor of BCR/ABL fusion gene, this agent has proven highly effective in patients with CML.And now, imatinib has been recommended as the first line thrapy for CML in NCCN.Although imatinib is effective for CML, part of patients become imatinib-resistant in early or the course of treatment.The resistant mechanism of imatinib may be various, but now, resistance to imatinib from BCR/ABL gene amplification, leading to overexpression of BCR/ABL protein, or point mutations in the BCR/ABL gene are deemed to be the most important mechanism of imatinib-resistant. However, sevaral researchs on imatinib-resistant K562cell lines found that:they did not discover BCR/ABL gene amplification, leading to overexpression of BCR/ABL protein, or point mutations in the BCR/ABL gene in these imatinib-resistant K562cell lines, on the contrary, they found the high level of Lyn which was one major member of SFK family.Therefore, this study test the expression of Lyn in76CML patients with clinical characteristics, clinical effect and chromosome abnomality to explore the significance of Lyn in imatinib-resustant CML.Methods:Western blotting was used to observe the expression of Lyn in bone marrow mononuclear cells of76patients with CML and10normal human being.Gene expression of BCR/ABL was detected by real-time PCR.Cytogenetic data were obtained from10of them by R band karyotypic analtysis.Using the data SPSS17.0statistical software t test (or nonparametric test)、Spearman rank correlation (or Pearson correlation), P<0.05showed a statistically significant.Results:1、Lyn and imatinib-resistant Lyn expression in imatinib-resistant patients was significantly higher than normal human being、 newly diagnosed patients and effective patients (P=0.0025、P=0.0014、 P=0.0001).However, there was no statistically significant difference between newly diagnosed patients、effective patientsnormal and human being (P=0.310、P=0.2355)2、BCR/ABL gene quantitative analysis We detected the expreession of BCR/ABL gene in newly diagnosed patients、effective patients and imatinib-resistant patients, the results showed that BCR/ABL expression in imatinib-resistant patients and newly diagnosed patientswas was significantly higher than effective patients (P=0.035、 P=0.026)3、Lyn and BCR/ABL gene Correlation between Lyn and BCR/ABL was analyzed, there was no obvious relation between Lyn and BCR/ABL (P=0.2780)4、Lyn and clinical characteristic All the CML patients and normal human being expreessed Lyn.There was no significant relationship with median age, gender, median hemoglobin, and median platets level, percentage of peripheral blasts, size of spleen (P=0.07、 P=0.068、P=0.869、P=0.968、P=0.873、P=0.647).The Lyn expression of the mean leucocyte above100×109/L at the first visit was obviously higher (P=0.0465)5、Lyn and chromosomes There was1case with chromosome abnormality in t (6;22) and t (2;9) in10imatinib-resistsnt CML patients, coexisting with Ph chromosome.The rest of9cases only existed Ph chromosome.There was no relationship between Ph chromosome and other clonal chromosome abnormality except Ph chromosome (P=0.487)Conclutions:1、CML and normal human being all expressed Lyn.2、Lyn was overexpressed in imatinib-resistant CML.3、There was no obvious relation with median age, gender, median hemoglobin, and median platets level, percentage of peripheral blasts, size of spleen.The increased Lyn expression had closely relationship with WBC count level.4、There was no correlation between Lyn and BCR/ABL.5、Lyn was not relevant to karyotype.更多还原