中性粒细胞与再生障碍性贫血免疫抑制治疗疗效关系的研究

发布时间：2018-06-24 09:07

本文选题：贫血 + 再生障碍性　；参考：《北京协和医学院》2017年博士论文

【摘要】：目的探讨重型/极重型再生障碍性贫血(SAA/VSAA)患者免疫抑制治疗(IST)前应用粒细胞集落刺激因子(G-CSF)治疗的中性粒细胞反应(ANC反应),对于IST早期疗效的预测价值。方法回顾性分析我中心呢2005年9月至2011年11月间接受一线IST治疗,并于IST前接受G-CSF治疗的共125例SAA/VSAA患者临床资料。G-CSF治疗第n天内最大△ANC≥0.5×109/L定义为G-CSF治疗有ANC反应,0.5×109/L定义为无反应。首先分别总结IST前应用G-CSF治疗有ANC反应组和无ANC反应组的IST近期疗效,并对二者进行相关性分析。进而为细化、优化这一预测指标,分别分析G-CSF治疗第5天、7天、10天、13天、14天、16天和21天有无ANC反应与IST早期疗效的关系,寻找预测IST早期疗效的最佳ANC反应时间。进而,绘制G-CSF治疗后10天内ANC增高程度(△ANC)预测IST疗效的ROC曲线,探索G-CSF治疗的ANC反应预测IST早期疗效的最佳△ANC界限值。结果IST前接受G-CSF治疗,获得ANC反应组IST后3个月血液学反应(HR)为49%,6个月HR为61.2%,均显著高于无ANC反应组(3个月HR 28.9%,P=0.023;6个月HR40.8%,P=0.026)。G-CSF治疗5天,7天内ANC反应尚不能提示IST后3个月及6个月HR,而以G-CSF治疗10 d内△ANC≥0.5×109/L作为ANC反应界值预测IST后3个月及6个月疗效最佳,ROC曲线下面积最大,且与更长时间应用G-CSF治疗ANC反应来预测IST后HR的效能接近。G-CSF治疗10 d内获得ANC反应为IST后3个月疗效的独立预后因素(P=0.004),但并非IST后6个月疗效的独立预后因素。G-CSF治疗ANC反应组患者5年总生存率显著高于无反应组[(92.8±4.0)%对(69.5±6.5)%,P=0.025]。结论IST前G-CSF治疗的ANC反应可提示骨髓残存造血,是方便、实用的预测IST早期疗效及长期生存的指标。目的分析不同中性粒细胞计数(ANC)阈值的极重型再生障碍性贫血(VSAA)患者接受一线免疫抑制治疗(IST)的早期血液学反应(HR)和长期生存情况。方法回顾性分析我中心2006年1月至2011年10月期间连续收治的145例接受一线r-ATG联合环孢素IST的VSAA患者临床资料。评估不同ANC阈值下VSAA患者的早期死亡、早期HR、长期生存情况,并以受试者操作特征曲线(ROC曲线)法优化ANC预测VSAA患者HR的临界值。并进行VSAA患者HR及生存相关因素分析。结果VSAA患者IST后3个月HR率为35.9%,6个月HR率为46.9%,5年OS率为75.0±3.7%,均明显劣于SAA(IST后3个月HR率为65.5%,P=0.000;6个月HR 率为 74.5%,P=0.000;5 年 OS 率为 92.5±2.6%,P=0.000)。且 VSAA 早期死亡率高达8.3%(SAA患者仅为2.7%,P=0.062)。设定VSAAIST前ANC阈值分别≤0.15×109/L、≤0.10×109/L和≤0.05×109/L,相应群组患者早期死亡率分别为9.2%(12/130)、9.9%(11/111)和 13.4%(11/82),即 12 例早期死亡的 VSAA 中有 11 例(91.7%)IST 前 ANC≤0.05 X 109/L;IST 后 3 个月 HR 率分别为 33.8%(44/130)、27.9%(31/111)和 22.0%(18/82);6 个月 HR 率分别为 43.8%(57/130)、39.6%(44/111)和 34.1%(28/82);5 年 OS 率分别为 73.1 ±4.0%、70.3±4.4%和62.5±5.4%;5 年 EFS 率分别为 48.3±4.5%、45.1 ±4.8%和 42.3±5.5%。以 0.05×109/L为ANC界值预测VSAA患者IST后6个月HR和12个月CR+GPR最佳,预测IST后6个月HR灵敏度为58.8%、特异度为70.1%;预测12个月良好HR灵敏度60.9%、特异度72.4%。ANC≤0.05×109/L的VSAA患者早期死亡率为11/82(13.4%),占VSAA 早期死亡患者的 91.7%(11/12);IST 后 3 个月 HR 率为 18/82(22.0%);6个月 HR 率为 28/82(34.1%);5 年 OS 率为(62.5±5.4)%;5 年 EFS 率为(42.3±5.5)%。结论ANC≤0.05×109/L的VSAA患者接受一线IST早期死亡率高、疗效差。目的ASXL1基因突变常见于各髓系肿瘤和骨髓衰竭性疾病的恶性转化,且为发生白血病转化和不良预后的相关因素。本研究旨在探讨ASXL1基因突变对人白血病细胞功能的影响及其作用的分子生物学机制,为ASXL1基因突变在髓系肿瘤性疾病中的作用提供更多依据,为个体化和靶向治疗提供更多的线索。方法应用CRISPR/Cas9基因编辑系统构建ASXL1敲除的U937人白血病细胞系。应用流式细胞术进行GFP阳性单个细胞分选并扩增为单个细胞来源的克隆。Sanger测序检测各细胞系ASXL1基因突变情况,筛选出含ASXL1杂合及纯和突变的克隆细胞系。应用瑞士吉姆萨染色分析细胞形态,G带进行细胞染色体核型分析,应用5-氟尿嘧啶诱导细胞凋亡,研究其对各细胞系增殖抑制和凋亡诱导情况,应用PMA诱导细胞分化并比较ASXL1突变细胞系与野生型分化情况异同,应用流式细胞术测定细胞周期、细胞凋亡和细胞分化等。进行RNA测序筛选在ASXL1突变克隆中差异性表达的基因,并进而进行信号通路分析、基因集分析等,研究ASXL1突变对下游基因表达的影响,应用逆转录定量PCR和蛋白印迹法验证差异性表达基因。结果应用CRISPR/Cas9基因编辑技术结合单个细胞分选技术构建单个细胞来源的人U937细胞系,共获得56个野生型细胞系(ASXL1基因野生型的U937单个细胞来源细胞系),另外17个突变型细胞系(6个含ASXL1杂合突变和11个含ASXL1纯和突变),突变细胞系均含有共同的c.594insA(Ser199Glufsx55)位点突变,此突变基因能够编辑提前终止编码的截短蛋白。其中3个ASXL1杂合突变克隆(MT1,MT2和MT3),3个ASXL1纯和突变克隆(MT11,MT12和MT13),2个转染后的ASXL1野生型克隆(WT1和WT2),以及未经转染的亲代细胞系WTblk用于进一步细胞功能及RNA测序等实验。整体来看,各个单细胞克隆之间异质性较大,但野生组与ASXL1杂合突变组、ASXL1纯和突变组,在细胞增殖,凋亡,细胞周期分布,5-氟尿嘧啶诱导的细胞增殖抑制和诱导细胞凋亡方面并无显著差异。而ASXL1杂合突变组和ASXL1纯和突变组相对于野生组均表现出对于PMA诱导的单核细胞向吞噬细胞分化能力显著降低,表现为PMA诱导分化后96小时,CD11阳性细胞比例及CD11中位荧光指数均显著低于野生组。RNA测序及信号通路和基因集分析显示,ASAL1杂合及纯和突变克隆,髓系分化相关基因通路整体表达下降,其中与髓系分化相关的基因CYBB和CLEC5A表达水平显著降低。同时,与细胞存活相关的基因集包括干扰素a反应通路、MYC靶基因通路和STAT3靶基因通路等广泛下调。此外,染色体核型分析显示相对于未经转染的亲代细胞系WTblk,转染后的ASXL1野生型克隆并未出现额外的染色体核型异常,而ASXL1突变克隆不同程度得获得额外染色体异常,在4个检测的ASXL1突变克隆中有3个获得11号染色体长臂缺失。结论ASXL1基因突变通过下调与髓系分化密切相关的CYBB和CLEC5A等基因干扰髓系分化。此外,ASXL1突变广泛干扰与细胞生存相关的基因通路,并增加人白血病细胞的遗传不稳定性。我们应用CRISPR/Cas9基因编辑技术建立的ASXL1突变型人U937白血病细胞系可作为研究ASXL1作用分子机制的模型,并未未来研究ASXL1相关的髓系恶性肿瘤相关分子机制的研究提供可能。
[Abstract]:Objective to explore the predictive value of neutrophils response (ANC reaction) for the early application of granulocyte colony-stimulating factor (G-CSF) therapy for severe / severe aplastic anemia (SAA/VSAA) patients with granulocyte colony-stimulating factor (G-CSF) treatment. Methods a retrospective analysis was made to receive first-line IST treatment between September 2005 and November 2011. The clinical data of 125 cases of SAA/VSAA patients treated with G-CSF before IST,.G-CSF was defined as G-CSF therapy with ANC reaction in n days, and 0.5 x 109/L was defined as no response. First, the short-term efficacy of G-CSF treatment group and non reactive group before IST was summarized, and the correlation analysis of two cases was analyzed. And then to optimize this prediction index, we analyzed the relationship between G-CSF treatment for fifth days, 7 days, 10 days, 13 days, 14 days, 16 days and 21 days, in order to find the best time to predict the early effect of IST, to find the best time to predict the early effect of IST, and then to draw the ROC curve of ANC increase (delta ANC) in the 10 days after G-CSF treatment (delta ANC) to predict the IST curative effect, and explore G-C. The ANC reaction of the SF treatment predicted the best Delta ANC limit for the early effect of IST. Results before IST, G-CSF treatment was accepted, and the hematological reaction (HR) was 49% in ANC reaction group and 61.2% for HR in 6 months after IST, which was significantly higher than that without ANC reaction group (3 months HR 28.9%, 6 months%, 6 months). T 3 months and 6 months HR, and G-CSF treatment 10 d Delta ANC > 0.5 x 109/L as ANC reaction to predict IST 3 months and 6 months of the best effect, the maximum area under the ROC curve, and longer time application of G-CSF ANC reaction to predict the effectiveness of IST after IST treatment 10 The post factor (P=0.004), but not the independent prognostic factor of the 6 months after IST, the total 5 year survival rate in the ANC reaction group was significantly higher than that in the non reactive group [(92.8 + 4)% to (69.5 + 6.5)%. P=0.025]. conclusion ANC reaction before IST G-CSF therapy could suggest bone marrow remnant hematopoiesis, which is convenient and practical to predict the early curative effect and long-term survival of IST. Objective. Objective to analyze the early hematological response (HR) and long-term survival of extremely severe aplastic anemia (VSAA) patients with different neutrophils counts (ANC) threshold. Methods a retrospective analysis of 145 consecutive patients receiving first-line r-ATG associated cyclosporine from January 2006 to October 2011 The clinical data of VSAA patients with vegetarian IST. Evaluate the early death of VSAA patients under different ANC thresholds, early HR, long-term survival, and optimize the critical value of ANC prediction of HR in VSAA patients by the operator's operation characteristic curve (ROC curve) method. The HR and survival correlation factors of VSAA patients were analyzed. Results the rate of VSAA patients was 35.9%, 6 months after 3 months of VSAA. The rate of OS was 46.9%, and the rate of 5 years was 75 + 3.7%. The rate of HR was significantly inferior to that of SAA (3 months after IST, HR rate was 65.5%, P=0.000; HR rate in 6 months was 74.5%, P=0.000; OS rate of 5 years was 92.5 + 2.6%, P=0.000). And the early VSAA mortality was up to 8.3% (SAA patients only 2.7%, P=0.062). The early mortality of the corresponding group was 9.2% (12/130), 9.9% (11/111) and 13.4% (11/82), that is, 11 cases (91.7%) of 12 early deaths (91.7%) IST ANC < 0.05 X 109/L, and HR rates of 33.8% (44/130), 27.9% (31/111) and 22%, respectively, 3 months after IST, 6, 27.9% and 22% respectively. % (28/82); the rate of OS in 5 years is 73.1 + 4%, 70.3 + 4.4% and 62.5 + 5.4%, and 5 years EFS rate is 48.3 + 4.5%, 45.1 + 4.8% and 42.3 + 109/L as ANC boundary value prediction for VSAA patients IST. .9%, the early mortality of VSAA patients with specific degree of 72.4%.ANC < 0.05 x 109/L was 11/82 (13.4%), accounting for 91.7% (11/12) of early death in VSAA; HR rate in 3 months after IST was 18/82 (22%); 6 months HR rate was 28/82 (34.1%); 5 years was (62.5 + 5.4)%, and the rate was (42.3 + 5.5)% in 5 years. The early mortality of IST is high and the curative effect is poor. Objective ASXL1 gene mutation is common in the malignant transformation of various myeloid tumors and bone marrow failure diseases, and it is the related factors of leukemia transformation and poor prognosis. The aim of this study is to explore the effect of ASXL1 gene mutation on the function of human leukemia cells and the molecular biological mechanism of ASXL1 basis. It provides more evidence for the role of mutation in myeloid tumor diseases and provides more clues for individualized and targeted therapy. Methods the CRISPR/Cas9 gene editing system was used to construct a ASXL1 knockout U937 human leukemia cell line. Flow cytometry was used to select and amplify the single cell of GFP positive single cells to be cloned.Sange R sequencing was used to detect the mutation of ASXL1 gene in each cell line, screening the clones containing ASXL1 heterozygous and pure and mutated cells. Using Swiss Giemsa staining to analyze cell morphology, G band for cell chromosome karyotype analysis, 5- fluorouracil induced apoptosis, to study the proliferation inhibition and apoptosis induction of each cell line, and to apply PMA inducement. Differentiation and comparison of ASXL1 mutant cell lines and wild type differentiation, cell cycle, cell apoptosis and cell differentiation were measured by flow cytometry. RNA sequencing was used to screen differentially expressed genes in ASXL1 mutant clones, and then signal pathway analysis, gene set analysis, and so on, to study the ASXL1 mutation on the lower swimming base. The differential expression gene was verified by reverse transcriptase PCR and Western blot. Results the human U937 cell line of single cell source was constructed by CRISPR/Cas9 gene editing technique and single cell sorting technique, and 56 wild type cell lines (U937 single cell source cell lines of ASXL1 gene wild type) were obtained. 17 mutant cell lines (6 ASXL1 heterozygous mutations and 11 ASXL1 pure and mutants) with a common c.594insA (Ser199Glufsx55) mutation, which can edit the truncated protein of the early termination code. 3 ASXL1 heterozygous clones (MT1, MT2 and MT3), 3 ASXL1 pure and mutant clones (MT11, MT12, and clones) MT13), 2 transfected ASXL1 wild type clones (WT1 and WT2) and untransfected parent cell line WTblk were used for further cell function and RNA sequencing experiments. Overall, the heterogeneity of each single cell clone was larger, but the wild group and the ASXL1 heterozygous mutation group, ASXL1 pure and mutant group, in cell proliferation, apoptosis, cell cycle distribution, There was no significant difference in the inhibition of cell proliferation induced by 5- fluorouracil and the induction of apoptosis. The ASXL1 heterozygous mutation group and the ASXL1 pure and mutation group showed a significant reduction in the differentiation ability of the monocyte to the phagocytic cells induced by PMA, 96 hours after PMA induced differentiation, the proportion of CD11 positive cells and CD11. The median fluorescence index was significantly lower than that in the wild group.RNA sequencing and signal transduction and gene set analysis. ASAL1 heterozygosity and pure and mutant clones, the overall expression of the myeloid differentiation related gene pathway decreased, and the expression level of CYBB and CLEC5A related to myeloid differentiation was significantly reduced. The perturbed a pathway, the MYC target gene pathway and the STAT3 target gene pathway were widely downregulated. In addition, the chromosome karyotype analysis showed that the ASXL1 wild type clones had no extra chromosome karyotype abnormalities compared to the untransfected parent cell line WTblk, and the ASXL1 mutant clones had to obtain extra chromosomal abnormalities in different degrees, at 4 3 of the ASXL1 mutant clones detected to be detected in the long arm deletion of chromosome 11. Conclusion the ASXL1 gene mutation interferes with the myeloid differentiation by reducing the CYBB and CLEC5A genes closely related to the myeloid differentiation. Furthermore, the ASXL1 mutation widely interferes with the gene pathway associated with cell survival and increases the genetic instability of the human leukemia cells. The ASXL1 mutant human U937 leukemia cell line established by CRISPR/Cas9 gene editing technique can be used as a model to study the molecular mechanism of ASXL1, and it is not possible to study the molecular mechanism of ASXL1 related myeloid malignancies in the future.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R556.5

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