【作者】 耿莉；

【导师】 王学谦； 李玉明；

【作者基本信息】 天津医科大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 目的:真性红细胞增多症（Polycythemia vera,PV）是克隆性的造血干细胞疾病,属于慢性骨髓增殖性疾病。PV的临床特征是红细胞不同程度地增多,同时可伴有血小板、白细胞增多及脾肿大,动、静脉血栓发生率高。少部分病例可进展为骨髓纤维化或转为急性髓细胞白血病。PV患者对促红细胞生成素（erythropoietin,EPO）高度敏感,体外可以在缺乏细胞因子的情况下形成红系克隆（内源性红系克隆增生,Endogenous erythroid colonies,EEC）,但其机制不清。最近,在多数PV患者中发现了Janus激酶2（JAK2）基因的一个体细胞点突变,突变是由鸟嘌呤（G）变成了胸腺嘧啶（T）,使得位于JH2假激酶区617位氨基酸错义编码(缬氨酸→苯丙氨酸,JAK2^V617F),导致激酶活性增高。JAK2^V617F突变造成患者EPO超敏、红细胞生成增多,导致真性红细胞增多症的发生。本研究旨在检测PV患者JAK2^V617F突变的发生率及其特异性,并分析其突变状态与临床、实验室特征的关系,以期为PV的诊断、预后判断、治疗提供依据。方法:采用等位基因特异性聚合酶链反应（Allele-specific PCR,AS-PCR）方法检测JAK2^V617F突变,包括26例已确诊的PV患者、10例继发性红细胞增多症患者、5例Ph+慢性粒细胞白血病（Chronic myelocytic leukemia,CML）患者及5例健康志愿者,并经DNA测序方法验证。通过聚合酶链反应-限制性片段长度多态性分析（restriction fragment length polymorphism,RFLP）,检测JAK2^V617F突变患者的突变状态,统计分析纯合子及杂合子突变与PV临床及实验室特征的关系。结果:26例PV患者中,有24例（92.3%）出现了JAK2^V617F突变,10例继发性红细胞增多症患者、5例Ph⁺CML患者及5例健康志愿者均为野生型JAK2基因。24例发生JAK2基因突变的患者中,7例为纯合子突变,17例为杂合子。比较2组临床及实验室特征,发现纯合子组初诊时外周血血红蛋白（Hb）水平高于杂合子组（224.29±6.24g/L vs.210.29±9.12g/L）,两组比较差异有统计学意义（p=0.001）。纯合子组初诊时血清乳酸脱氢酶（LDH）亦高于杂合子组（488.00±149.09 IU/L vs.339.47±129.10 IU/L）,两组比较差异有统计学意义（p=0.023）。而2组病例在发病年龄、性别、生存期、初诊时白细胞及血小板计数、肝脾肿大发生率、皮肤瘙痒症、血栓性疾病发生率、转化为骨髓纤维化比例等方面的差异无统计学意义。结论:1.JAK2^V617F突变在PV患者中高表达,该突变在继发性红细胞增多症、Ph⁺CML患者中未检出。2.JAk2^V617F突变可能成为PV的分子诊断标准。3.JAK2^V617F突变纯合子较杂合子初诊时Hb、LDH水平高。更多还原

【Abstract】 Objective:Polycythemia vera（PV）,one of the chronic myeloproliferative disorders,is a clonal haematopoietic stem cell malignancy.The clinical course of patients with PV is characterized by the presence of polycythemia diversely associated with thrombocytosis,leukocytosis and splenomegaly,and an increased rate of arterial and venouse thrombosis.Less frequently,the disease might develop myelofibrosis or transforms to acute myeloid leukemia.In polycythemia vera,the mechanisms leading to erythropoietin hypersensitivity and in vitro production of erythroid colonies in the absence of cytokines（endogenous erythroid colonies,EEC） are still unknown.Recently,a somatic point mutation of the Janus kinase 2（JAK2）gene was identified in majority of patients with PV.This point mutation is a G to T transversion,resulting in a nonsynonymous amino acid substitution at position 617（valine to phenylalanine）located in the JH2 pseudo-kinase,leading to increased kinase activity.JAK2^V617Finduces erythropoietin hypersensitivity and erythrocytosis,results in PV.The aim of this study was to investigate the incidence and specificity of JAK2^V617Fmutation in patients with PV,to analyze the correlations between JAK2^V617Fmutational status and clinical,laboratory features,so that we can provide evidencese for the diagnosis,prognosis and management of patients with PV.Methods:By AS-PCR,the expression of JAK2^V617Fof 26 patients with a diagnosed PV,10 patients with secondary erythrocytosis,5 patients with Ph⁺ CML and 5 healthy controls was assayed.And the results were confirmed by sequence analysis.PCR-RFLP was performed to identify the mutation status of JAK2^V617FA statistical analysis between JAK2^V617Fheterozygotes and homozygotes was performed associations with clinical and laboratory characteristic.Results:The JAK2^V617Fmutant allele was detected in 24 of the 26 patients （92.3%）.10 patients with secondary erythrocytosis,5 patients with Ph⁺ CML and 5 healthy controls had the wild-type JAK2.Seven of patients with JAK2^V617Fmutant allele were homozygotes,17 patients were heterozygotes.A comparison between the homozygote patients and the heterozygote patients associations with clinical and laboratory revealed significantly higher hemoglobin level at the time of diagnosis in the former group（224.29±6.24 g/L vs.210.29±9.12g/L,p= 0.001）.Similarly,a significantly higher LDH level were finded in patients with homozygous JAK2^V617F,compared with those with the heterozygous mutation.However,a statistical comparison between JAK2^V617Fheterozygotes and homozygotes did not reveal any significant associations with regard to age,gender,survival time,leukocyte or platelet count at the time of diagnosis,or the incidences of splenomegaly or hepatomegaly, pruritus,thrombosis and fibrotic transformation.Conclusions:1.JAK2^V617Fmutation was highly expressed in PV.2.JAK2^V617Fmutation maybe becomes the molecular diagnostic criteria of PV.3.The hemoglobin level and LDH level at the time of diagnosis were significantly higher in patients with homozygous JAK2^V617F,compared with those with the heterozygous mutation.更多还原