骨髓增殖性肿瘤JAK2V617F、CALR及MPL基因检测与并发血栓性疾病关系的研究

发布时间：2018-05-25 05:03

本文选题：骨髓增殖性肿瘤 + JAK2V617F　；参考：《山东大学》2016年博士论文

【摘要】：骨髓增殖性肿瘤(MPN)是骨髓持续克隆增殖性造血干细胞疾病。经典的BCR-ABL阴性MPN包括真性红细胞增多症(PV)、原发性血小板增多症(ET)和原发性骨髓纤维化(PMF)。血栓栓塞是MPN患者死亡的主要原因,并严重影响患者的生存质量,早期及时的发现血栓并相应干预将大大的降低患者的死亡率。JAK2V617F基因突变为经典的BCR-ABL阴性MPN的特征性改变,除此以外,还发现有CALR及MPL基因突变。JAK2V617F、CALR及MPL基因突变与血栓性疾病的关系国内虽有研究,但均为回顾性。本研究检测了MPN患者的JAK2V617F、CALR及MPL基因突变率,跟踪2年观察其血栓性疾病的发生率,并通过检测MPN患者单个核细胞的pSTAT3、TNF-α mRNA和IL-6 mRNA的表达,对MPN并发血栓的发病机制做了进一步的深入研究。第一部分MPN患者JAK2V617F、CALR和MPL基因突变率及其临床特征研究(基线研究)[研究目的]检测MPN患者JAK2V617F、CALR和MPL基因突变率并调查与血栓形成相关的临床特征。[研究方法](1)研究对象共3组,病例组为2013年9月至2014年4月确诊为MPN且没有血栓并发症的患者,共68例(病例组,n=68)；血栓组为同期确诊为动脉或静脉血栓形成者,但无血液疾病,共65例(血栓组,n=65)；对照组为同期健康体检者或血液内科病情稳定的非MPN患者,没有血栓并发症,共68例(对照组,n=68)。(2)记录所有受试者的年龄、性别、居住地、职业等一般资料及既往史、吸烟史、酗酒史等,采集所有研究对象的血压值、空腹血糖值、血低密度脂蛋白浓度、高密度脂蛋白浓度、胆固醇浓度值、身体质量指数等与血栓形成有关的危险因子及凝血常规和血常规等。(3)采集所有研究对象隔夜空腹肘静脉血各3 ml用于检测JAK2V617F、CALR、 MPL基因突变。采集所有研究对象隔夜空腹肘静脉血足量用于检测空腹血糖、血脂谱、凝血常规和血常规等指标。(4)用人JAK2V617F基因变异检测试剂盒提取血细胞中的基因组DNA,采用LightCycler 480荧光PCR基因扩增检测仪进行PCR扩增,用AB13730型基因分析仪测序,输出结果。[结果](1)对三组研究对象进行JAK2V617F基因突变进行检测,68例MPN患者有46例发生JAK2V617F基因突变,突变率为67.65%。其中ET患者24例,突变率为64.86%,PV患者19例,突变率为82.61%,IMF患者3例,突变率为37.5%。68例对照组中JAK2V617F突变阳性为1例,突变率为1.47%。MPN患者组JAK2V617F突变率显著高于对照组(P0.05)。65例血栓组中JAK2V617F突变阳性为8例,突变率为12.31%。MPN患者组JAK2V617F突变率显著高于血栓组(P0.05)。血栓组JAK2V617F突变率显著高于对照组(P0.05)。对三组研究对象进行CALR基因突变进行检测。68例MPN患者有12例发生CALR基因突变,突变率为17.65%,其中包括ET患者10例,PV患者0例,PMF患者2例。68例对照组中CALR突变阳性为0例。MPN患者组与对照组比较P0.05,差异有统计学意义。65例血栓组CALR突变阳性为0例。MPN患者组与血栓组比较P0.05,差异有统计学意义。对三组研究对象进行MPL基因突变进行检测。68例MPN患者有3例发生MPL基因突变,突变率为4.41%,其中包括ET患者2例,PV患者0例,IMF患者1例。68例对照组中MPL突变阳性为0例。MPN患者组与对照组比较P0.05,差异有统计学意义。65例血栓组MPL突变阳性为0例。MPN患者组与血栓组比较P0.05,差异有统计学意义。MPN患者中三种基因全阴性的7例,占10.29%,三种突变没有同时出现。(2)对病例组JAK2V617F阳性患者与JAK2V617F阴性患者间血栓风险因子的比较,JAK2V617F阳性MPN患者与JAK2V617F阴性MPN患者间的收缩压、舒张压、空腹血糖值和血脂谱值等均无显著性统计学差异(P0.05)。对病例组CALR阳性患者与CALR阴性患者间血栓风险因子的比较,除了空腹血糖外,两组患者间的收缩压、舒张压、LDL、HDL、BMI、胆固醇等血栓风险因子均无显著性统计学差异。比较病例组MPL阳性患者与MPL阴性患者间血栓风险因子的比较,两组患者间的空腹血糖、收缩压、舒张压、LDL、HDL、BMI、胆固醇等血栓风险因子均无显著性统计学差异。(3)病例组PT值[(16.94±2.07)s]显著高于对照组[(12.26±±0.96)s](P0.05)：病例组APTT值[(59.26±12.38)s]显著高于对照组[(30.73±3.13)s](P0.05)；病例组TT值[(16.58±1.75)s]显著高于对照组[(12.53±1.07)s](P0.05);病例组FIB值[(3.02±±0.58)g/L]显著低于对照组[(3.36±±0.49)g/L](P0.05)。病例组中JAK2V617F突变阳性患者的PT为(17.87+1.56)s]显著高于JAK2V617F突变阴性患者[(15.00±1.60)s(P0.05);JAK2V617F突变阳性患者的APTT[(64.22±10.82)s]显著高于JAK2V617F突变阴性患者[(48.91±8.49)s (P0.05); JAK2V617F突变阳性患者的TT[(16.98±1.642)s]显著高于JAK2V617F突变阴性患者[(15.78±1.72)s(P0.05)。病例组中的CALR突变阴性患者与CARL突变阳性患者间的PT、APTT、TT、FIB均无显著性统计学差异,且病例组中的MPL突变阴性患者与MPL突变阳性患者间的PT、APTT、TT、FIB均无显著性统计学差异。[结论及意义]MPN患者有显著较高的JAK2V617F、CALR和MPL基因突变率;MPN患者中JAK2V617F基因突变阳性与阴性患者间的已知血栓形成风险因子无显著差异;MPN患者中JAK2V617F基因突变阳性患者凝血功能较阴性患者显著异常。第二部分MPN患者2年内血栓事件发生的随访及其机制研究[研究目的]探讨MPN患者JAK2V617F、CALR及MPL基因突变与血栓性疾病发生的相关性及其机制研究。[研究方法](1)研究对象为第一部分的病例组和对照组。自入组之日起,电话或门诊随访所有研究对象至少每月两次,询问并记录所有研究对象的详细病史,尤其是与血栓事件及血液病发生相关的病史,以及血压值、体重及期间的血液检测指标等。共随访2年,没有失访者,最晚者随访至2016年4月。(2)分离MPN组患者外周血单个核细胞,提取细胞质总蛋白,免疫印迹法检测pSTAT3蛋白表达,提取外周血单个核细胞mRNA, RQ-PCR检测TNF-α mRNA. IL-6 mRNA的表达。[结果](1)追踪患者2年发生血栓性疾病情况。68例MPN患者中有26例患者发生血栓,血栓发生率为38.24%。对照组有2例发生血栓,发生率为2.94%,病例组的血栓发生率显著高于对照组(P0.05)。病例组JAK2V617F阳性患者中血栓患者为47.83%(22/46),非血栓患者为52.17%(24/46)；病例组JAK2V617F阴性患者中血栓患者为18.18%(4/22),非血栓患者为81.82%(18/22), JAK2V617F阳性MPN患者的血栓发生率显著高于JAK2V617F阴性MPN患者(P=0.019)。CALR和MPL基因突变阳性MPN患者的血栓发生率均为0。(2)病例组PT值[(17.04±2.15)s]显著高于对照组[(12.18±0.88)s](P0.05)；病例组APTT值[(58.15±12.49)s]显著高于对照组[(30.87±3.00)s](P0.05)；病例组TT值[(17.59±2.81)s]显著高于对照组[(12.47±1.04)s](P0.05)；病例组FIB值[(3.05+0.56)g/L]显著低于对照组[(3.33±0.44)g/L](P0.05)。(3)ET患者中血栓患者PLT值[(840.15±48.60)×109/L],显著高于ET患者中非血栓患者[(809.46±36.98)×109/L](P0.05)。病例组中血栓患者TT为(18.85±2.72)s显著高于非血栓患者(16.81±2.60)s(P0.05)。(4)对病例组患者与对照组研究对象间血栓风险因子的比较：随访2年后,病例组患者与对照组间的收缩压值(P0.05)、舒张压值(P0.05)、空腹血糖值(P0.05)、血脂谱值(P0.05)均无显著性统计学差异。(5)对病例组血栓患者与非血栓患者间血栓风险因子的比较：随访2年后,病例组中血栓患者与非血栓患者间的收缩压值(P0.05)、舒张压值(P0.05)空腹血糖值(P0.05)、血脂谱值(P0.05)均无显著性统计学差异。(6)MPN患者血栓并发症与外周血单个核细胞中P-STAT3的表达间的关系：与JAK2V617F突变阴性的MPN患者相比,JAK2V617F突变阳性的患者中P-STAT3蛋白灰度值显著较高(P0.05)；与无血栓并发症的MPN患者相比,发生血栓并发症的MPN患者有显著较高的P-STAT5蛋白表达灰度值(P0.05)。JAK2V617F突变阳性且并发血栓的MPN患者的P-STAT3蛋白表达灰度值显著较高(P0.05)。(7)MPN患者血栓并发症与外周血单个核细胞中IL-6 mRNA的表达间的关系：与JAK2V617F突变阴性的MPN患者相比,JAK2V617F突变阳性的患者中IL-6mRNA相对值显著较高(P0.05)；与无血栓并发症的MPN患者相比,发生血栓并发症的MPN患者有显著较高的IL-6 mRNA相对值(P0.05);JAK2V617F突变阳性且并发血栓的MPN患者的IL-6 mRNA相对值显著较高(P0.05)。[结论及意义]MPN患者中JAK2V617F基因突变阳性患者易发血栓事件；JAK2V617F基因突变阳性是MPN患者发生血栓事件的原因；MPN患者JAK2V617F基因突变阳性可能通过某种信号转导路径影响致凝血常规检测结果显著异常,最终导致血栓事件发生；IL-6相关的、经STAT3活化的JAK-STAT信号路径可能是JAK2V617F突变阳性的MPN患者血栓并发症的机制之一。本研究的创新点1、本研究检测了所有研究对象的JAK2V617F、CALR及MPL基因的突变率,通过设立对照组和2年的跟踪随访,对MPN患者血栓发生与JAK2V617F、 CALR及MPL基因突变阳性间的关系做了前瞻性的研究,发现MPN患者中JAK2V617F基因突变阳性患者易发血栓事件,CALR及MPL基因突变突变没有增加MPN患者的血栓发生率。2、通过检测MPN患者单个核细胞的pSTAT3、TNF-α mRNA、IL-6 mRNA的表达,提示IL-6相关的、经STAT3活化的JAK-STAT信号路径可能是JAK2V617F突变阳性的MPN患者发生血栓并发症的机制之一。
[Abstract]:Myeloproliferative tumor (MPN) is a persistent and proliferative hematopoietic stem cell disease of the bone marrow. The classic BCR-ABL negative MPN includes true erythrocytosis (PV), primary thrombocythemia (ET) and primary myelofibrosis (PMF). Thromboembolism is the main cause of death in MPN patients and seriously affects the patient's quality of life, early and timely. The discovery of thrombus and corresponding intervention will greatly reduce the death rate of the patient's.JAK2V617F gene to the classic BCR-ABL negative MPN. In addition, there are also CALR and MPL gene mutations.JAK2V617F, and the relationship between the CALR and MPL gene mutations and thrombotic diseases in China, although they are all reviewed. This study detected M The mutation rate of JAK2V617F, CALR and MPL gene in PN patients was followed up for 2 years to observe the incidence of thrombotic diseases, and the pathogenesis of MPN concurrent thrombus was further studied by detecting the expression of pSTAT3, TNF- a mRNA and IL-6 mRNA in the mononuclear cells of the patients with MPN. The clinical feature study (baseline study) [Objective] to detect the mutation rate of JAK2V617F, CALR and MPL genes in MPN patients and to investigate the clinical features associated with thrombosis. [research methods] (1) a total of 3 subjects were studied in a case group of 68 patients (case group, n=68), and 68 cases (case group, n=68). The group was diagnosed as an arterial or venous thrombosis in the same period, but there were no blood diseases, 65 cases (thrombus group, n=65), and the control group was a healthy physical examination or a non MPN patient with stable condition in the blood department. There was no thrombus complication, 68 cases (control group, n=68). (2) record all the subjects' age, sex, residence, occupation and so on. History, smoking history, drinking history, etc., collected all the subjects' blood pressure, fasting blood glucose, HDL concentration, HDL concentration, cholesterol concentration, body mass index and other risk factors related to thrombosis, clotting routine and blood routine. (3) all subjects were collected on the overnight empty abdominal elbow vein blood of 3. Ml was used to detect JAK2V617F, CALR, and MPL mutations. All subjects were used to measure the blood glucose, blood lipid spectrum, blood clotting routine and blood routine. (4) the gene group DNA in blood cells was extracted by the JAK2V617F gene mutation detection kit, and the LightCycler 480 fluorescent PCR gene amplification detector was used. PCR amplification, AB13730 gene analyzer sequencing, output results. [results] (1) three groups of subjects were detected JAK2V617F gene mutation, 68 cases of MPN patients with JAK2V617F gene mutation, the mutation rate was 67.65%. of 24 cases of ET patients, the mutation rate was 64.86%, PV patients 19 cases, the mutation rate was 82.61%, IMF patients 3 cases, mutation In the control group, the rate of JAK2V617F mutation was 1 cases in the control group, and the mutation rate was higher than that of the control group, the JAK2V617F mutation rate was significantly higher than that of the control group (P0.05), the JAK2V617F mutation was 8 in the.65 thrombus group, and the mutation rate was significantly higher than that in the blood thrombus group (P0.05) in the group of 12.31%.MPN patients. The JAK2V617F mutation rate in the thrombus group was significantly higher than that in the group of 37.5%.68. Control group (P0.05). CALR gene mutations in three groups of subjects were detected in 12 cases of.68 cases MPN mutation, the mutation rate was 17.65%, including 10 cases of ET patients, 0 cases of PV patients, 2 cases of PMF patients in.68 case control group, 0 cases of.MPN patients were compared with the control group, the difference was statistically significant. The CALR mutation in the thrombus group was 0 cases of.MPN patients and the thrombus group was P0.05, the difference was statistically significant. 3 cases of MPN patients in the three groups of subjects were detected with MPL gene mutation, the mutation rate was 4.41%, including 2 cases of ET patients, 0 cases of PV patients, 1.68 case control group of IMF patients, 0 MPL mutations were 0. Cases of.MPN patients were compared with the control group P0.05, the difference was statistically significant in.65 cases, MPL mutation was positive in 0 cases of.MPN patients and P0.05 in thrombus group. The difference was statistically significant in 7 cases of all negative three genes in.MPN patients, accounting for 10.29%, and three kinds of mutations did not appear simultaneously. (2) the case group JAK2V617F positive patients and JAK2V617F negative There was no significant difference in systolic pressure, diastolic blood pressure, fasting blood glucose and blood lipid profiles between JAK2V617F positive MPN patients and JAK2V617F negative MPN patients (P0.05). Two groups of patients with CALR positive patients and CALR negative patients were compared with fasting blood glucose. There was no significant difference in systolic blood pressure, diastolic pressure, LDL, HDL, BMI, cholesterol and other thrombotic risk factors. Compared with the thrombus risk factors between the MPL positive patients and the MPL negative patients, there was no significant difference between the two groups of fasting blood glucose, systolic pressure, diastolic pressure, LDL, HDL, BMI, cholesterol and other thrombotic risk factors. (3) the PT value of the case group [(16.94 + 2.07) s] was significantly higher than that of the control group (12.26 + 0.96) s] (P0.05): the APTT value of the case group was significantly higher than that of the control group [(30.73 + 3.13) s] (P0.05), and the TT value [(16.58 + 1.75) s] of the case group was significantly higher than that of the control group [12.53 + 1.07) s] (P0.05). .36 + 0.49) g/L] (P0.05). The PT of JAK2V617F mutation positive patients in the case group was significantly higher than that of JAK2V617F mutation negative patients [(15 + 1.60) s (P0.05), JAK2V617F mutation positive patients' APTT[(64.22 + 10.82) was significantly higher than that of the negative patients [48.91 + 8.49); 16. 98 + 1.642) s] was significantly higher than that of JAK2V617F mutant negative patients [(15.78 + 1.72) s (P0.05). There was no significant difference in PT, APTT, TT, FIB between the CALR mutation negative patients and the CARL positive patients in the case group, and there was no significant difference between the MPL and the MPL mutations. [Conclusion and significance]MPN patients have significantly higher JAK2V617F, CALR and MPL mutation rates; there is no significant difference in the known thromboformation risk factors between the JAK2V617F gene mutation positive and negative patients in MPN patients, and there is a significant abnormal coagulation function in patients with JAK2V617F gene mutation in MPN patients. Second partial MPN patients 2 Follow up and mechanism study of thrombus events during the year [Objective] to investigate the correlation and mechanism of JAK2V617F, CALR, and MPL mutations in MPN patients with thrombotic diseases. [1] the research object is the first part of the case group and the control group. From the day of entry, all the subjects are followed up by telephone or outpatient service. Two times a month, we asked and recorded the detailed history of all the subjects, especially the history of thrombosis and hematological diseases, and the blood pressure, weight and blood test indexes during the period of 2 years. No missing persons were followed up to April 2016. (2) isolated peripheral blood mononuclear cells from the MPN group were isolated and the cells were extracted. Total protein, immunoblotting was used to detect the expression of pSTAT3 protein, mRNA in peripheral blood mononuclear cells, and the expression of TNF- alpha mRNA. IL-6 mRNA in peripheral blood. [results] (1) 26 cases of patients with thrombotic disease in 2 years were traced and 26 patients had thrombus. The incidence of thrombosis was 2 cases in the control group of 38.24%. control, and the incidence was 2. .94%, the incidence of thrombus in the case group was significantly higher than that in the control group (P0.05). The thrombus patients in the case group JAK2V617F positive patients were 47.83% (22/46), the non thrombus patients were 52.17% (24/46); the thrombus patients in the case group JAK2V617F negative patients were 18.18% (4/22), the non thrombus patients were 81.82% (18/22), and the thrombus incidence was significant in the JAK2V617F positive MPN patients. The incidence of thrombus in the.CALR and MPL positive MPN patients with JAK2V617F negative MPN (P=0.019) was 0. (2) ((17.04 + 2.15) s] significantly higher than that of the control group [(12.18 + 0.88) s] (P0.05)), and the APTT value [(58.15 + 12.49)) of the case group was significantly higher than that of the Group [(30.87 + 3)]. Higher than the control group [(12.47 + 1.04) s] (P0.05)), the FIB value of the case group [(3.05+0.56) g/L] was significantly lower than that of the control group [(3.33 + 0.44) g/L] (P0.05). (3) the thrombotic patients in ET patients [(840.15 + 48.60) * 109/L], significantly higher than those in the ET patients [(809.46 + 36.98) * 109/L]]. In patients with non thrombus (16.81 + 2.60) s (P0.05). (4) a comparison of thrombus risk factors between the patients and the control group: after 2 years of follow-up, the systolic pressure (P0.05), diastolic blood pressure (P0.05), fasting blood glucose (P0.05) and blood lipid (P0.05) between the patients and the control group had no significant difference. (5) thrombosis in the case group. Comparison of thrombus risk factors between patients and non thrombotic patients: after 2 years of follow-up, the systolic pressure value (P0.05), diastolic blood pressure (P0.05) fasting blood glucose (P0.05) and blood lipid (P0.05) in patients with thrombus were not significantly different in the case group. (6) thrombus complication in MPN patients and P-STAT3 in peripheral blood mononuclear cells Relationship between expression: the gray value of P-STAT3 protein was significantly higher in patients with JAK2V617F mutations than in patients with negative JAK2V617F mutations (P0.05). Compared with MPN patients without thrombotic complications, the MPN patients with thrombotic complications had a significantly higher level of P-STAT5 protein expression (P0.05).JAK2V617F mutation positive and concurrence. The gray value of P-STAT3 protein expression in MPN patients with thrombus was significantly higher (P0.05). (7) the relationship between thrombus complications and the expression of IL-6 mRNA in peripheral blood mononuclear cells in MPN patients: the relative value of IL-6mRNA was significantly higher in patients with positive JAK2V617F mutations than in MPN patients with negative JAK2V617F mutations (P0.05); M with thrombotic complications Compared to patients with PN, the MPN patients with thrombotic complications had a significant higher relative value of IL-6 mRNA (P0.05), and the IL-6 mRNA relative value of the MPN patients with positive JAK2V617F mutation and thrombus was significantly higher (P0.05). [Conclusion and]MPN patients with JAK2V617F genes mutation positive patients were prone to thrombosis; The cause of thrombus events in patients; the JAK2V617F gene mutation in MPN patients may be significantly abnormal through some signal transduction pathway and eventually leads to thrombus events; IL-6 related, STAT3 activated JAK-STAT signaling pathway may be a thrombotic complication of MPN patients with positive JAK2V617F mutation. One of the innovative points of this study. 1. This study examined the mutation rates of the JAK2V617F, CALR and MPL genes in all the subjects. By setting up a control group and a follow-up follow-up of 2 years, a prospective study was made on the relationship between thrombosis and the positive mutations of the JAK2V617F, CALR and MPL genes in MPN patients, and the JAK2V617F gene process in MPN patients was found. The mutation of CALR and MPL gene mutations did not increase the rate of thrombosis in MPN patients.2. The expression of pSTAT3, TNF- a mRNA, and IL-6 mRNA in the mononuclear cells of patients with MPN suggested IL-6 related. One of the mechanisms of the disease.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R733.3

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