【作者】 孙国锋；

【导师】 潘崚；

【作者基本信息】 河北医科大学 ， 内科学， 2007， 硕士

【摘要】 目的:真性红细胞增多症(PV)、原发性血小板增多症(ET)和特发性骨髓纤维化(IMF)组成了经典的BCR/ABL阴性的慢性骨髓增殖性疾病(cMPDS)。与BCR/ABL阳性的CML不同,在PV、ET和IMF患者没有发现可重复性染色体易位,也无特效治疗药物。近期的一些研究发现BCR/ABL阴性的cMPDS患者中广泛存在一种特异性基因突变: JAK2基因的第1849位点发生G→T突变,导致JAK2蛋白第617位的缬氨酸被苯丙氨酸代替(简称为JAK2 V617F)。约90%的PV患者存在该突变,并且在其他BCR/ABL阴性的cMPDS患者如ET和IMF中也有相当的比例,而在其他血液病中罕见此突变。ETS2(人红血细胞增多症病毒致癌基因同源体2)是ETS转录因子家族的一员,ETS2基因定位于21号染色体21q22.3,编码分子量为56-kDa的蛋白质,它在肿瘤发生、骨骼发育和免疫应答中扮演重要角色。近期有研究表明在BCR/ABL阴性的cMPDs中, JAK2 V617F点突变阳性患者的ETS2 mRNA表达水平高于阴性者。本研究旨在探讨BCR/ABL阴性的cMPDs中JAK2 V617F点突变的发生率、ETS2 mRNA表达情况及其与cMPDs分类、疗效、并发症发生率及预后之间的关系。通过JAK2 V617F突变率与ETS2 mRNA表达情况的相关性研究,了解JAK2 V617F点突变与ETS2 mRNA表达水平的异常在cMPDs发生发展中的作用机制,期望为寻找针对性治疗方法奠定基础。方法:1本实验研究对象包括62例BCR/ABL阴性的cMPDs患者[26例PV、26例ET、9例IMF、1例慢性中性粒细胞性白血病(CNL)];20例慢性粒细胞性白血病(CML)患者、10例急性白血病(AL)患者,15名健康志愿者。62例cMPDS患者为研究组、其他患者和正常人为对照组。所有病例符合WHO诊断标准。2从各组患者及健康志愿者的骨髓或血液中提取单个核细胞,再从其中提取总RNA及DNA。3用半定量逆转录-聚合酶链反应(RT-PCR)技术检测各组ETS2 mRNA的表达水平。4用等位基因特异性聚合酶链反应(AS-PCR)筛选各组JAK2V617F点突变,根据检查结果将62例cMPDs患者分为突变阳性组及阴性组。全部阳性标本和随机抽取的部分阴性对照标本,经PCR、产物纯化后,进行基因测序以验证突变结果。5分别观察和比较各组病例的临床特征,包括:性别、发病年龄、临床症状及体征、血象、骨髓像、并发症、疗效和预后等。6统计学处理:采用单因素方差分析比较ETS2 mRNA在MPD组、急性白血病组、慢性粒细胞白血病组与正常对照组中的表达水平有无差异;卡方检验分析JAK2 V617F点突变率在各组的差异。ETS2 mRNA表达水平在cMPDs JAK2 V617F点突变阳性组与阴性组比用t检验。所有数据用SPSS11.0统计软件分析处理。结果:1 62例BCR/ABL阴性的cMPDs患者中,共有44例患者检测到JAK2 V617F点突变,包括23/26(88.46%)例PV,15/26(57.69%)例ET,5/9(55.56%)例IMF。1/1(100.00%)例CNL。在CML、AL及健康志愿者中未检测到JAK2 V617F点突变。2 ETS2 mRNA表达水平在cMPDs组(平均值0.2876,阳性率81.96%)、CML组(平均值0.4133,阳性率85.0%)和AL(平均值0.5320,阳性率80.0%)组均显著高于健康志愿者(平均值0.106,阳性率46.7%)。cMPDs组、CML组、AL组之间比较,ETS2 mRNA表达水平均值无明显差别。3在cMPDs组JAK2 V617F点突变阳性的病例中ETS2 mRNA表达水平(平均值0.323,阳性率86.0%)明显高于突变阴性病例(平均值0.203,阳性率72.2%),差别有显著意义,P=0.022。4临床特征对比:PV患者中JAK2 V617F点突变阳性组的外周血白细胞计数显著高于阴性组(平均值18.2×109/L vs 7.6×109/L,P=0.035),血小板计数显著高于阴性组(平均值479×109/L vs 277×109/L,P=0.025)。ET患者中JAK2 V617F点突变阳性组的血红蛋白显著高于阴性组(平均值146g/L vs 122g/L, P=0.001),白细胞计数显著高于阴性组(平均值14.6×109/L vs 10.9×109/L, P=0.044)。在IMF患者中JAK2 V617F点突变阳性组与阴性组之间的临床特征无显著性差异,P>0.05。结论:1大部分cMPDs患者存在JAK2 V617F点突变(PV 88.46%、ET 57.69%、IMF 55.56%),提示JAK2 V617F点突变所产生的激活作用是PV、ET、IMF的重要发病机制。2在JAK2 V617F点突变阳性的cMPDs患者中,ETS2 mRNA的表达水平高于JAK2 V617F点突变阴性的cMPDS患者。更多还原

【Abstract】 Objectives: The classical BCR/ABL negative chronic myeloproliferative diseases (cMPDs) consist of polycythemia vera (PV),essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF). Differing from BCR/ABL positive chronic myelogenous leukemia (CML), neither a repeatable chromosome translocation was discovered in PV, ET and IMF patients nor was an effective medicine developed. Recently several studies discovered a highly pathogenic characteristic gene mutation, a G to T change in the 1849th codon of JAK2 gene, and the amino-isovaleric acid is accordingly replaced byβ-phenyl-α-aminopropionic acid in the 617th of JAK2 tyrosine kinase. This mutation is defined as JAK2 V617F(or JAK2 val617phe)point mutation and has been detected in up to 90% of PV patients and in a sizeable proportion of patients with other cMPDs such as ET and IMF, but rarely be found in other hematological disease. This finding is a landmark for understanding the molecular pathogenesis of BCR/ABL negative cMPDs.On the orther hand, erythroblastosis virus oncogene homolog 2 (ETS2) is a pro-oncogene, located in human chromosomal region 21q22.3, expresses in various tissues,including blood,breast and prostate. This gene encodes a 56 kD protein that is phosphorylated by a Ca2+-dependent mitogenic signal process. ETS2 protein is a member of the ETS family of transcription factors, involved in the regulation of cellular proliferation and differentiation and may play a critical role in T-cell activation and cytokines production. It had been reported that ETS2 gene was associated with the growth and invasion of breast carcinoma cells and was required to maintain the transformed state for human prostate cancer cells. It is also reported that ETS2 mRNA was over-expressed in cMPDs. However, it is not clear whether there is a relationship between the mutation of JAK2 V617F and ETS2 mRNA overexpression and whether they both influence the progression of BCR/ABL negative cMPDs. The present study is thus designed to examine the somatic JAK2 V617F mutation rate and measure the expression level of ETS2 mRNA in cMPDs,acute leukemia and normal control, to make a further study on the molecular biological pathogenesis of BCR/ABL negative cMPDs, and to try to provide theoretical basis for developing possible targeted medicines for the treatment of JAK2 V617F mutation positive cMPDs.Methods:1 Heparinized peripheral blood or bone marrow was obtained after informed consent from 62 BCR/ABL negative cMPDs patients (26 PV cases, 26 ET cases,9 IMF cases and 1 CNL case), 20 CML cases, 10 acute leukemia (AL) cases and 15 healthy volunteers. The 62 of BCR/ABL negative cMPDs patients were designed as research group and the others were control group. All these patients’diagnosis fit the WHO criteria made in 2000.2 Total RNA and DNA was extracted from samples of the peripheral blood mononuclear cells (PBMNCs) or bone marrow mononuclear cells (BMMNCs) taken from patients in each group or healthy volunteers.3 The expression level of ETS2 mRNA was measured by RT-PCR method, which was established and widely used in our lab.4 JAK2 V617F mutation was detected by AS-PCR method and confirmed by direct DNA sequencing. Then the BCR/ABL negative cMPDs patients were divided into two subgroups: JAK2 V617F mutation positive group and mutation negative group.5 Patients’clinical characteristics including age, gender, clinical symptoms and signs, hemograms, bone marrow pictures, bone marrow pathological features and the reaction to conventional therapy and prognosis of each patient in different subgroup were observed and compared.6 Statistical analyses: Most statistic analysis was performed using the SPSS 11.0 version for windows. The statistically significant meaning of the difference in JAK2 V617F mutation rate was analyzed by Chi-square analysis. The statistically significant meaning of the difference in ETS2 mRNA expression level between JAK2 V617F mutation positive group and mutation negative group was analyzed by t-test. The results were shown as mean±SD(X—±S).Results:1 The JAK2 V617F point mutation was detected in 44 BCR/ABL negative MPD patients, including 23/26 (88.46%)PV patients,15/26(57.69%)ET patients, 5/9(55.56%)IMF patients and 1/1 (100% )CNL patient. While the somatic JAK2 V617F mutation was not detected either in the 20 CML patients, 10 AL patients or the 15 health volunteers.2 The relative expression level of ETS2 mRNA in BCR/ABL negative cMPDs group (mean value 0.2876, positive rate 81.96%), or in CML group (mean value 0.4133, positive rate 85.0%), or in AL group (mean value 0.5320, positive rate 80.0%) is statistically significant higher than that in health volunteer group (mean value 0.106 , positive rate 46.7%). P<0.05.3 The relative expression level of ETS2 mRNA in JAK2 V617F mutation positive group (mean value 0.323 , positive rate 86.0%) is statistically significant higher than that in negative group (mean value 0.203 , positive rate 72.2%). P=0.022.4 The comparison of clinical characteristics: The peripheral white blood cell counts (mean 18.2×109/L) and platelets counts (mean 479×109/L) in JAK2 V617F mutation positive PV patients were statistically significant higher than that (7.6×109/L and 277×109/L, respectively) in JAK2 V617F negative PV patients (P=0.035 and P=0.025,respectively) at diagnosis. Compared with the mutation negative ET patients, the mutation positive ET patients had statistically significant higher hemoglobin (122g/L vs 146g/L, P=0.001) and peripheral white blood cell counts (10.9×109/L vs 14.6×109/L, P=0.044) as well as a higher complication rate (P=0.034). But no statistically significant difference in the clinical data was found between JAK2 V617F positive and negative IMF patients (P>0.05).Conclusions:1 A somatic JAK2 V617F point mutation was detected in a sizeable proportion of BCR/ABL fusion gene negative cMPDs patients: the mutation rate was 88.46% in PV, 57.69% in ET and 55.56% in IMF patients. This mutation was not detected in AL patients, CML patients or healthy volunteers.2 The expression level of ETS2 mRNA in JAK2 V617F mutation positive group is statistically significant higher than that in negative group.更多还原