MPL及CALR基因突变对骨髓增殖性肿瘤患者中性粒细胞碱性磷酸酶表达的作用研究

发布时间：2018-04-28 05:16

本文选题：骨髓增殖性肿瘤 + NAP　；参考：《山西医科大学》2017年硕士论文

【摘要】：目的:骨髓增殖性肿瘤(MPN)是一组以成熟髓系细胞恶性增殖为主要特征的克隆性造血干细胞疾病。JAK2 V617F、MPL、CALR基因突变为MPN的主要分子病因,这些突变均发生于髓系干细胞阶段,进而导致髓系干细胞以下阶段各髓系细胞受累。中性粒细胞碱性磷酸酶(NAP)是成熟中性粒细胞的一种表达产物,常作为反映中性粒细胞成熟程度的重要指标。前期研究已证实JAK2 V617F基因突变可使受累的中性粒细胞NAP表达增高,临床上患者表现为NAP积分值增高。本研究将从临床水平和细胞水平分别探讨MPL、CALR基因突变体对受累中性粒细胞NAP表达的影响。方法:1、临床水平比较JAK2 V617F、MPL、CALR突变体对骨髓增殖性肿瘤患者中性粒细胞碱性磷酸酶表达变化的影响收集2014年7月至2016年7月在山西医科大学第二医院血液科就诊伴JAK2V617F、MPL或CALR基因突变阳性MPN患者,包括真性红细胞增多症(PV)、原发性血小板增多症(ET)、原发性骨髓纤维化(PMF)。回顾性收集所有患者初诊时外周血NAP积分数据(NAP积分值均来自同一实验室),对各突变组患者NAP积分值进行统计学分析。2、细胞水平研究MPL、CALR突变体对中性粒细胞碱性磷酸酶表达的作用构建MPL、CALR各突变基因及其对照基因慢病毒表达载体,通过慢病毒表达系统将MPL及CALR各突变基因载体及对照基因载体转导于急性早幼粒细胞性白血病细胞株(NB4),建立稳定表达各载体的NB4细胞模型;联合应用粒细胞集落刺激因子(G-CSF)与全反式维甲酸(ATRA)诱导培养各载体NB4细胞模型,收集48h、72h、96h、120h、144h各诱导时间点的各组细胞(5×105个),采用磷酸对硝基苯酯(p NPP)法检测各组细胞中性粒细胞碱性磷酸酶表达量并进行统计学分析。结果:1、JAK2V617F、MPL、CALR基因突变阳性骨髓增殖性肿瘤患者初诊时中性粒细胞碱性磷酸酶(NAP)积分值比较分析收集初诊MPN患者共125例,其中ET患者75例,PMF患者19例,PV患者31例。75例ET患者中,JAK2V617F阳性51例、MPL阳性4例、CALR阳性20例,JAK2V617F阳性患者NAP积分均值高于正常范围,MPL、CALR阳性患者NAP积分均值则低于正常范围,JAK2V617F阳性患者与CALR(P0.01)或MPL(P=0.001)阳性患者之间NAP积分均有显著统计学差异,MPL阳性患者与CALR阳性患者之间NAP积分无统计学差异(P=0.245);19例PMF患者中,JAK2V617F阳性13例、MPL阳性2例、CALR阳性4例,JAK2V617F阳性患者NAP积分均值高于正常范围,MPL、CALR阳性患者NAP积分均值则低于正常范围,JAK2V617F阳性患者与CALR(P=0.003)或MPL(P=0.027)阳性患者之间NAP积分均有显著统计学差异,MPL阳性患者与CALR阳性患者之间NAP积分无统计学差异(P=0.814);31例PV患者均为JAK2V617F阳性,其NAP积分均值高于正常范围。2、稳定表达MPL各突变基因及对照基因NB4细胞模型经诱导后NAP表达的比较分析收集48h、72h、96h、120h、144h各诱导时间点各组细胞(5×105个),检测其NAP表达,结果显示:随着诱导分化时间的延长,NB4组、MSCV组、MPLWT组NAP表达呈逐渐增高,MPLW515L组与MPLA497-L498LVIAins4组表达变化不明显;在诱导分化96h后,MPLW515L组、MPLA497-L498LVIAins4组分别与NB4组、MSCV组、MPLWT组有显著统计学差异(P0.05)。3、稳定表达CALR各突变基因及对照基因NB4细胞模型经诱导后NAP表达的比较分析收集48h、72h、96h、120h、144h各诱导时间点各组细胞(5×105个),检测其NAP表达,结果显示:随着诱导分化时间的延长,NB4组、MSCV组、CALRWT组NAP表达呈逐渐增高,CALRtype1组与CALRtype2组表达变化不明显;在诱导分化96h后,CALRtype1组、CALRtype2分别与NB4组、MSCV组、CALRWT组有显著统计学差异(P0.05)。结论:JAK2 V617F可致其受累中性粒细胞的碱性磷酸酶表达增高,而MPL、CALR突变体则使受累中性粒细胞碱性磷酸酶表达降低,提示虽JAK2 V617F、MPL、CALR突变体均可导致MPN,但它们在发病机制及临床表型方面却存在异质性。
[Abstract]:Objective: bone marrow proliferative tumor (MPN) is a group of major molecular causes of.JAK2 V617F, MPL, CALR gene mutation, which are the main characteristics of malignant proliferation of mature myeloid cells. These mutations occur at the stage of myeloid stem cells, which lead to the involvement of the medullary cells in the following stages of myeloid stem cells. Cell alkaline phosphatase (NAP) is an expression product of mature neutrophils. It is often used as an important indicator to reflect the maturity of neutrophils. Earlier studies have confirmed that the JAK2 V617F gene mutation can increase the NAP expression of the affected neutrophils, and the clinical patients show a higher NAP score. This study will be from the clinical level and the cells. The effects of MPL, CALR gene mutants on the expression of NAP in infected neutrophils were examined. Methods: 1, the effects of JAK2 V617F, MPL, CALR mutants on the expression of neutrophils alkaline phosphatase in patients with myeloproliferative tumors were compared at the clinical level and collected from July 2014 to July 2016 at the Department of Hematology at the Department of Hematology, Shanxi Medical University MPN patients with JAK2V617F, MPL, or CALR mutations, including genuine erythrocytosis (PV), primary thrombocytopenia (ET), and primary myelofibrosis (PMF). A retrospective collection of NAP integral data of peripheral blood in all patients (all NAP scores from the same laboratory) was collected, and the statistical credits of the NAP integral values of the patients in each mutation group were counted. Analysis of.2, cell level of MPL, CALR mutant on the expression of neutrophil alkaline phosphatase, construct MPL, CALR mutation gene and its control gene Lentivirus Expression Vector, transduce MPL and CALR mutant gene vectors and control gene vectors to acute promyelocytic leukemia cell line (NB4) through the lentivirus expression system. The NB4 cell model for the stable expression of each carrier was established. Combined with granulocyte colony stimulating factor (G-CSF) and all trans retinoic acid (ATRA), each carrier NB4 cell model was induced, and the cells of 48h, 72h, 96h, 120h, 144H each induction time point were collected (5 x 105), and the neutrophils were detected by phosphoric acid to nitrobenzene (P NPP) method. The expression of alkaline phosphatase and statistical analysis. Results: 1, JAK2V617F, MPL, CALR gene mutation positive myeloproliferative tumor patients at first diagnosis of neutrophils alkaline phosphatase (NAP) integral value analysis collected 125 cases of first diagnosed MPN patients, of which 75 cases of ET patients, PMF patients 19 cases, PV patients in.75 cases ET patients, JAK2V617F positive 5 1 cases, 4 cases of MPL positive and 20 cases of CALR positive, the mean value of NAP integral in JAK2V617F positive patients is higher than that of normal range. The mean value of NAP integral in MPL and CALR positive patients is lower than that in the normal range, and there are significant differences between JAK2V617F positive patients and CALR (P0.01) or MPL (P=0.001) positive patients. There were no statistical differences (P=0.245). In 19 cases of PMF, there were 13 cases of JAK2V617F positive, 2 cases of MPL positive, 4 cases of CALR positive, and the mean value of NAP integral in JAK2V617F positive patients was higher than that of normal range. The NAP integral of MPL and CALR positive patients was lower than normal range. There was no statistical difference between MPL positive patients and CALR positive patients (P=0.814). 31 cases of PV patients were JAK2V617F positive, the mean value of NAP integral was higher than that of normal range.2, and the stable expression of MPL mutation genes and the control gene NB4 cell model were compared and analyzed for NAP expression after induction. The expression of NAP was detected in each group of cells (5 x 105) at the guiding time point. The results showed that the expression of NAP in group NB4, MSCV and MPLWT increased gradually with the prolongation of induced differentiation time, and the expression of NAP in group MPLW515L and MPLA497-L498LVIAins4 group was not obvious. After inducing differentiated 96h, the MPLW515L group and MPLA497-L498LVIAins4 group were respectively with NB4 group, MSCV group, and group. There was a significant statistical difference (P0.05).3. A stable expression of CALR mutation genes and a control gene NB4 cell model after induction of NAP expression was compared and analyzed to collect 48h, 72h, 96h, 120h, 144H in each group (5 x 105) to detect its NAP expression. The expression of CALRtype1 and CALRtype2 was not obvious. After the induction of 96h, group CALRtype1 and CALRtype2 were significantly different from NB4 group, MSCV group and CALRWT group (P0.05). Conclusion: JAK2 V617F can increase the expression of alkaline phosphatase in the involved neutrophils, while MPL, the mutant makes the neutrophils involved. The decrease of alkaline phosphatase expression indicates that although JAK2 V617F, MPL and CALR mutants can lead to MPN, they are heterogeneous in pathogenesis and clinical phenotype.

【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.3

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