發(fā)布時(shí)間：2018-04-28 05:16

  本文選題：骨髓增殖性腫瘤 + NAP��； 參考：《山西醫(yī)科大學(xué)》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(xì)胞惡性增殖為主要特征的克隆性造血干細(xì)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(xì)胞階段,進(jìn)而導(dǎo)致髓系干細(xì)胞以下階段各髓系細(xì)胞受累。中性粒細(xì)胞堿性磷酸酶(NAP)是成熟中性粒細(xì)胞的一種表達(dá)產(chǎn)物,常作為反映中性粒細(xì)胞成熟程度的重要指標(biāo)。前期研究已證實(shí)JAK2 V617F基因突變可使受累的中性粒細(xì)胞NAP表達(dá)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(xì)胞水平分別探討MPL、CALR基因突變體對(duì)受累中性粒細(xì)胞NAP表達(dá)的影響。方法:1、臨床水平比較JAK2 V617F、MPL、CALR突變體對(duì)骨髓增殖性腫瘤患者中性粒細(xì)胞堿性磷酸酶表達(dá)變化的影響收集2014年7月至2016年7月在山西醫(yī)科大學(xué)第二醫(yī)院血液科就診伴JAK2V617F、MPL或CALR基因突變陽(yáng)性MPN患者,包括真性紅細(xì)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)�；仡櫺允占谢颊叱踉\時(shí)外周血NAP積分?jǐn)?shù)據(jù)(NAP積分值均來自同一實(shí)驗(yàn)室),對(duì)各突變組患者NAP積分值進(jìn)行統(tǒng)計(jì)學(xué)分析。2、細(xì)胞水平研究MPL、CALR突變體對(duì)中性粒細(xì)胞堿性磷酸酶表達(dá)的作用構(gòu)建MPL、CALR各突變基因及其對(duì)照基因慢病毒表達(dá)載體,通過慢病毒表達(dá)系統(tǒng)將MPL及CALR各突變基因載體及對(duì)照基因載體轉(zhuǎn)導(dǎo)于急性早幼粒細(xì)胞性白血病細(xì)胞株(NB4),建立穩(wěn)定表達(dá)各載體的NB4細(xì)胞模型;聯(lián)合應(yīng)用粒細(xì)胞集落刺激因子(G-CSF)與全反式維甲酸(ATRA)誘導(dǎo)培養(yǎng)各載體NB4細(xì)胞模型,收集48h、72h、96h、120h、144h各誘導(dǎo)時(shí)間點(diǎn)的各組細(xì)胞(5×105個(gè)),采用磷酸對(duì)硝基苯酯(p NPP)法檢測(cè)各組細(xì)胞中性粒細(xì)胞堿性磷酸酶表達(dá)量并進(jìn)行統(tǒng)計(jì)學(xué)分析。結(jié)果:1、JAK2V617F、MPL、CALR基因突變陽(yáng)性骨髓增殖性腫瘤患者初診時(shí)中性粒細(xì)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(yáng)性51例、MPL陽(yáng)性4例、CALR陽(yáng)性20例,JAK2V617F陽(yáng)性患者NAP積分均值高于正常范圍,MPL、CALR陽(yáng)性患者NAP積分均值則低于正常范圍,JAK2V617F陽(yáng)性患者與CALR(P0.01)或MPL(P=0.001)陽(yáng)性患者之間NAP積分均有顯著統(tǒng)計(jì)學(xué)差異,MPL陽(yáng)性患者與CALR陽(yáng)性患者之間NAP積分無統(tǒng)計(jì)學(xué)差異(P=0.245);19例PMF患者中,JAK2V617F陽(yáng)性13例、MPL陽(yáng)性2例、CALR陽(yáng)性4例,JAK2V617F陽(yáng)性患者NAP積分均值高于正常范圍,MPL、CALR陽(yáng)性患者NAP積分均值則低于正常范圍,JAK2V617F陽(yáng)性患者與CALR(P=0.003)或MPL(P=0.027)陽(yáng)性患者之間NAP積分均有顯著統(tǒng)計(jì)學(xué)差異,MPL陽(yáng)性患者與CALR陽(yáng)性患者之間NAP積分無統(tǒng)計(jì)學(xué)差異(P=0.814);31例PV患者均為JAK2V617F陽(yáng)性,其NAP積分均值高于正常范圍。2、穩(wěn)定表達(dá)MPL各突變基因及對(duì)照基因NB4細(xì)胞模型經(jīng)誘導(dǎo)后NAP表達(dá)的比較分析收集48h、72h、96h、120h、144h各誘導(dǎo)時(shí)間點(diǎn)各組細(xì)胞(5×105個(gè)),檢測(cè)其NAP表達(dá),結(jié)果顯示:隨著誘導(dǎo)分化時(shí)間的延長(zhǎng),NB4組、MSCV組、MPLWT組NAP表達(dá)呈逐漸增高,MPLW515L組與MPLA497-L498LVIAins4組表達(dá)變化不明顯;在誘導(dǎo)分化96h后,MPLW515L組、MPLA497-L498LVIAins4組分別與NB4組、MSCV組、MPLWT組有顯著統(tǒng)計(jì)學(xué)差異(P0.05)。3、穩(wěn)定表達(dá)CALR各突變基因及對(duì)照基因NB4細(xì)胞模型經(jīng)誘導(dǎo)后NAP表達(dá)的比較分析收集48h、72h、96h、120h、144h各誘導(dǎo)時(shí)間點(diǎn)各組細(xì)胞(5×105個(gè)),檢測(cè)其NAP表達(dá),結(jié)果顯示:隨著誘導(dǎo)分化時(shí)間的延長(zhǎng),NB4組、MSCV組、CALRWT組NAP表達(dá)呈逐漸增高,CALRtype1組與CALRtype2組表達(dá)變化不明顯;在誘導(dǎo)分化96h后,CALRtype1組、CALRtype2分別與NB4組、MSCV組、CALRWT組有顯著統(tǒng)計(jì)學(xué)差異(P0.05)。結(jié)論:JAK2 V617F可致其受累中性粒細(xì)胞的堿性磷酸酶表達(dá)增高,而MPL、CALR突變體則使受累中性粒細(xì)胞堿性磷酸酶表達(dá)降低,提示雖JAK2 V617F、MPL、CALR突變體均可導(dǎo)致MPN,但它們?cè)诎l(fā)病機(jī)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(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R733.3

【參考文獻(xiàn)】

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(xué)雜志;2006年10期

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本文編號(hào)：1813924