利用基因组编辑技术TALENs研究急性白血病DNMT3A突变的分子机制

发布时间：2018-05-21 21:07

本文选题：DNMT3A + R882H　；参考：《华中科技大学》2015年博士论文

【摘要】：目的：本研究旨在探讨成人急性淋巴细胞白血病(acute lymphoblastic leukemia, ALL)患者中DNA甲基转移酶3A (DNA methyltransferase, DNMT3A)突变发生的频率、与其他临床特征间的相关性以及对患者预后的影响。方法：收集57例急性淋巴细胞白血病患者的骨髓或外周血细胞,使用Ficoll梯度离心法分离单个核细胞,提取基因组DNA后采用PCR的方法对DNMT3A热点突变所在的第23号外显子进行扩增,PCR产物纯化后测序,与正常基因组序列比对筛查突变结果。通过本科室生物标本库获取57例患者相关临床特征信息并进行统计分析。结果：在57例ALL患者中,共检测出3例(5.3%)携带DNMT3A R882H突变。T-ALL患者中突变的发生率明显高于B-ALL患者(P=0.048)。年龄在40至60岁之间的患者与其他年龄组相比具有更高的突变发生率(P=-0.042)。在全部患者中和T-ALL中,携带DNMT3A突变的患者与野生型患者相比整体生存期较短(P=0.02和P=0.014)。结论：DNMT3A突变在ALL中发生率较低,突变患者其整体生存期明显缩短,提示该突变对成人ALL患者的危险分级和治疗具有重要的意义。目的：本研究旨在研究成人急性髓系白血病(acute myeloid leukemia, AML)患者中DNA甲基转移酶3A (DNA methyltransferase, DNMT3A)突变发生的频率、与其他临床特征间的相关性以及对患者预后的影响。方法：收集392例成人急性髓系白血病患者的骨髓或外周血细胞,使用Ficoll梯度离心法从中获取单个核细胞,提取基因组DNA后采用PCR的方法对DNMT3A热点突变所在的第23号外显子进行扩增,PCR产物纯化之后测序,最后与正常基因组序列比对判读突变结果。通过本科室生物标本库获取392例患者相关临床特征信息并进行统计分析。结果：在392例AML患者中,共检测出37例(9.44%)携带DNMT3A R882突变,其中DNMT3A R882H29例(78.38%),R882C8例(21.62%)。DNMT3A突变阳性患者与野生型患者相比,年龄明显偏大,但在性别、白细胞计数、血红蛋白水平、血小板计数和初诊时原始细胞比例等方面无明显差异。DNMT3A突变主要集中于M4和M5中,与NPM1突变和FLT3-ITD突变之间存在明显正相关性(P0.001)。在染色体核型分析中,DNMT3A突变主要集中在CN-AML中(83.78%,31/37,P0.001)以及中危组中(91.89%,34/37,P0.001)。生存分析显示,在整体AML和CN-AML患者中,DNMT3A突变阳性患者和野生型患者相比,OS(P=0.0218和P=0.0006)和EFS(P=0.0203和P=0.0006)均明显缩短。在CN-AML中,多因素分析结果显示DNMT3A突变可以作为一个独立的预后因素,提示较短的整体生存期(HR=1.986,95%CI1.207to3.269,P=0.007)。结论：DNMT3A突变在AML中发生频率较高,并且对患者预后具有不良影响,提示该突变对成人AML患者预后危险分级和治疗方案的选择具有重要的意义。目的：我们研究的目的在于利用TALENs技术构建一株携带DNMT3A突变的K562细胞系,并研究该突变对肿瘤细胞生物学效应的影响。方法：设计并使用快速模块组装法构建DNMT3A-TALEN质粒。TALEN质粒和供者质粒一起电转入K562细胞,之后用PCR的方法筛选携带DNMT3A R882H突变的克隆。用全外显子测序的方法检测DNMT3A-TALEN质粒可能存在的脱靶效应。蛋白质印迹技术检测DNMT3A和SLC7A11基因蛋白水平的表达变化。克隆形成试验和CFSE实验检测突变克隆的增殖能力。用流式细胞术检测细胞凋亡率。结果：本研究中,我们首次使用TALENs技术成功构建了一株携带DNMT3A R882H突变的K562细胞系。克隆形成试验和CFSE实验证实该突变可以明显促进细胞的增殖。表达谱芯片结果显示一些与GSH合成相关的关键基因表达明显升高,包括CTH,PSPH, PSAT1特别是SLC7A11(胱氨酸/谷氨酸转运体),从而导致突变克隆细胞内GSH含量升高。此外,检测细胞凋亡率结果显示突变克隆出现化疗抵抗,联合使用SSZ(柳氮磺胺吡啶)可以提高对化疗药物的敏感性。结论：本研究结果为DNMT3A R882H突变在AML发病机制中的作用提供了新的见解,DNMT3A R882H突变克隆细胞内GSH含量升高,出现化疗抵抗,联合使用SLC7A11的抑制剂SSZ可提高化疗药物敏感性,为携带DNMT3A R882H突变AML患者提供了新的诊疗思路。
[Abstract]:Objective: the purpose of this study was to investigate the frequency of DNA methyltransferase (DNA methyltransferase, DNMT3A) mutation in acute lymphoblastic leukemia (ALL) patients and the correlation with other clinical features and the effect on the prognosis of patients.
Methods: the bone marrow or peripheral blood cells of 57 patients with acute lymphoblastic leukemia were collected and the mononuclear cells were separated by Ficoll gradient centrifugation. After the extraction of genomic DNA, PCR was used to amplify the exon twenty-third of the DNMT3A hot spot mutation, and the PCR product was purified and sequenced and compared with the normal genome sequence to screen the mutant junctions. Results. The clinical characteristics of 57 patients were collected and analyzed statistically.
Results: among 57 patients with ALL, 3 (5.3%) patients carrying DNMT3A R882H mutation were significantly higher than those with B-ALL (P=0.048). Patients aged 40 to 60 had a higher mutation rate (P=-0.042) compared with other age groups. In all patients and T-ALL, patients carrying DNMT3A mutations Compared with the wild type patients, the overall survival time is shorter (P=0.02 and P=0.014). Conclusion: the incidence of DNMT3A mutation in ALL is low, and the overall survival time of the mutant patients is significantly shortened, suggesting that the mutation is of great significance for the risk classification and treatment of the adult ALL patients.
Objective: the purpose of this study was to study the frequency of DNA methyltransferase 3A (DNA methyltransferase, DNMT3A) mutation in patients with acute myeloid leukemia (AML), the correlation with other clinical features and the effect on the prognosis of patients. Methods: the bone marrow of 392 patients with adult acute myeloid leukemia was collected. Or peripheral blood cells, Ficoll gradient centrifugation was used to obtain mononuclear cells. After extracting genomic DNA, PCR was used to amplify the exon twenty-third of the DNMT3A hot spot mutation, and the PCR product was sequenced after purification. Finally, the mutation results were compared with the normal genome sequence. Through the biologic Specimen Bank of the undergraduate room, 392 cases were obtained. The clinical characteristics of patients were analyzed and statistically analyzed.
Results: of the 392 cases of AML, 37 cases (9.44%) were detected with DNMT3A R882 mutation, of which DNMT3A R882H29 (78.38%), R882C8 cases (21.62%).DNMT3A mutation positive patients were significantly older than those of wild type, but in sex, white cell count, blood erythrocyte level, platelet count and initial diagnosis of primitive cell ratio. There was no significant difference in.DNMT3A mutations in M4 and M5, and there was a significant positive correlation between NPM1 and FLT3-ITD mutations. In chromosome karyotype analysis, the DNMT3A mutation was mainly concentrated in CN-AML (83.78%, 31/37, P0.001) and in the middle risk group (91.89%, 34/37, P0.001). The OS (P=0.0218 and P=0.0006) and EFS (P=0.0203 and P=0.0006) were significantly shorter than those of the wild type patients. In CN-AML, the multivariate analysis showed that the DNMT3A mutation could be used as an independent prognostic factor, suggesting a shorter whole life period (HR =1.986,95%CI1.207to3.269, P=0.007).
Conclusion: DNMT3A mutations have a high frequency in AML and have adverse effects on the prognosis of the patients, suggesting that the mutation is of great significance for the prognostic risk classification and the choice of treatment options for adult AML patients.
Objective: the aim of this study was to construct a K562 cell line with DNMT3A mutation using TALENs technique and to study the effect of the mutation on the biological effects of tumor cells.
Methods: the DNMT3A-TALEN plasmid.TALEN plasmid was constructed and transformed into K562 cells with the donor plasmid, and then the DNMT3A R882H mutation was screened by PCR method. The possible Miss target effect of the DNMT3A-TALEN plasmid was detected by the exon sequencing method. The Western blot technique was used to detect DNMT3A. And the expression level of SLC7A11 gene protein. Clonogenic assay and CFSE assay were used to detect the proliferation ability of mutant clones. The apoptosis rate was detected by flow cytometry.
Results: in this study, we successfully constructed a K562 cell line carrying DNMT3A R882H mutation using TALENs technology. The clone formation test and CFSE experiment confirmed that the mutation could significantly promote the proliferation of cells. The expression spectrum chip results showed that the expression of some key genes related to GSH synthesis was significantly increased, including CTH, PSPH, PSAT. 1 in particular, SLC7A11 (cystine / glutamate transporter), resulting in increased GSH content in the mutant cloned cells. In addition, the detection of apoptosis results showed that the mutant clones were resistant to chemotherapy, and the combination of SSZ (sulfasalazine) could improve the sensitivity to chemotherapeutic drugs.
Conclusion: the results of this study provide a new insight into the role of DNMT3A R882H mutation in the pathogenesis of AML, the increase of GSH content in the DNMT3A R882H mutant cells and the emergence of chemotherapeutic resistance. The combined use of SLC7A11 inhibitor SSZ can improve the chemosensitivity of chemotherapy and provide a new diagnosis and treatment idea for the patients with DNMT3A R882H mutation AML.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R733.71

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