本文選題：骨髓增殖性腫瘤 + NAP�。� 參考：《山西醫(yī)科大學》2017年碩士論文

【摘要】：目的:骨髓增殖性腫瘤(MPN)是一組以成熟髓系細胞惡性增殖為主要特征的克隆性造血干細胞疾病。JAK2 V617F、MPL、CALR基因突變?yōu)镸PN的主要分子病因,這些突變均發(fā)生于髓系干細胞階段,進而導致髓系干細胞以下階段各髓系細胞受累。中性粒細胞堿性磷酸酶(NAP)是成熟中性粒細胞的一種表達產(chǎn)物,常作為反映中性粒細胞成熟程度的重要指標。前期研究已證實JAK2 V617F基因突變可使受累的中性粒細胞NAP表達增高,臨床上患者表現(xiàn)為NAP積分值增高。本研究將從臨床水平和細胞水平分別探討MPL、CALR基因突變體對受累中性粒細胞NAP表達的影響。方法:1、臨床水平比較JAK2 V617F、MPL、CALR突變體對骨髓增殖性腫瘤患者中性粒細胞堿性磷酸酶表達變化的影響收集2014年7月至2016年7月在山西醫(yī)科大學第二醫(yī)院血液科就診伴JAK2V617F、MPL或CALR基因突變陽性MPN患者,包括真性紅細胞增多癥(PV)、原發(fā)性血小板增多癥(ET)、原發(fā)性骨髓纖維化(PMF)。回顧性收集所有患者初診時外周血NAP積分數(shù)據(jù)(NAP積分值均來自同一實驗室),對各突變組患者NAP積分值進行統(tǒng)計學分析。2、細胞水平研究MPL、CALR突變體對中性粒細胞堿性磷酸酶表達的作用構(gòu)建MPL、CALR各突變基因及其對照基因慢病毒表達載體,通過慢病毒表達系統(tǒng)將MPL及CALR各突變基因載體及對照基因載體轉(zhuǎn)導于急性早幼粒細胞性白血病細胞株(NB4),建立穩(wěn)定表達各載體的NB4細胞模型;聯(lián)合應用粒細胞集落刺激因子(G-CSF)與全反式維甲酸(ATRA)誘導培養(yǎng)各載體NB4細胞模型,收集48h、72h、96h、120h、144h各誘導時間點的各組細胞(5×105個),采用磷酸對硝基苯酯(p NPP)法檢測各組細胞中性粒細胞堿性磷酸酶表達量并進行統(tǒng)計學分析。結(jié)果:1、JAK2V617F、MPL、CALR基因突變陽性骨髓增殖性腫瘤患者初診時中性粒細胞堿性磷酸酶(NAP)積分值比較分析收集初診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陽性51例、MPL陽性4例、CALR陽性20例,JAK2V617F陽性患者NAP積分均值高于正常范圍,MPL、CALR陽性患者NAP積分均值則低于正常范圍,JAK2V617F陽性患者與CALR(P0.01)或MPL(P=0.001)陽性患者之間NAP積分均有顯著統(tǒng)計學差異,MPL陽性患者與CALR陽性患者之間NAP積分無統(tǒng)計學差異(P=0.245);19例PMF患者中,JAK2V617F陽性13例、MPL陽性2例、CALR陽性4例,JAK2V617F陽性患者NAP積分均值高于正常范圍,MPL、CALR陽性患者NAP積分均值則低于正常范圍,JAK2V617F陽性患者與CALR(P=0.003)或MPL(P=0.027)陽性患者之間NAP積分均有顯著統(tǒng)計學差異,MPL陽性患者與CALR陽性患者之間NAP積分無統(tǒng)計學差異(P=0.814);31例PV患者均為JAK2V617F陽性,其NAP積分均值高于正常范圍。2、穩(wěn)定表達MPL各突變基因及對照基因NB4細胞模型經(jīng)誘導后NAP表達的比較分析收集48h、72h、96h、120h、144h各誘導時間點各組細胞(5×105個),檢測其NAP表達,結(jié)果顯示:隨著誘導分化時間的延長,NB4組、MSCV組、MPLWT組NAP表達呈逐漸增高,MPLW515L組與MPLA497-L498LVIAins4組表達變化不明顯;在誘導分化96h后,MPLW515L組、MPLA497-L498LVIAins4組分別與NB4組、MSCV組、MPLWT組有顯著統(tǒng)計學差異(P0.05)。3、穩(wěn)定表達CALR各突變基因及對照基因NB4細胞模型經(jīng)誘導后NAP表達的比較分析收集48h、72h、96h、120h、144h各誘導時間點各組細胞(5×105個),檢測其NAP表達,結(jié)果顯示:隨著誘導分化時間的延長,NB4組、MSCV組、CALRWT組NAP表達呈逐漸增高,CALRtype1組與CALRtype2組表達變化不明顯;在誘導分化96h后,CALRtype1組、CALRtype2分別與NB4組、MSCV組、CALRWT組有顯著統(tǒng)計學差異(P0.05)。結(jié)論:JAK2 V617F可致其受累中性粒細胞的堿性磷酸酶表達增高,而MPL、CALR突變體則使受累中性粒細胞堿性磷酸酶表達降低,提示雖JAK2 V617F、MPL、CALR突變體均可導致MPN,但它們在發(fā)病機制及臨床表型方面卻存在異質(zhì)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.

【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R733.3

【參考文獻】

相關(guān)期刊論文 前1條

1 宋君紅;張?zhí)K江;李建勇;;JAK2基因突變與慢性骨髓增殖性疾病[J];中華血液學雜志;2006年10期

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本文編號：1813924