本文選題：DNMT3A + R882H��； 參考：《華中科技大學》2015年博士論文

【摘要】：目的：本研究旨在探討成人急性淋巴細胞白血病(acute lymphoblastic leukemia, ALL)患者中DNA甲基轉移酶3A (DNA methyltransferase, DNMT3A)突變發(fā)生的頻率、與其他臨床特征間的相關性以及對患者預后的影響。 方法：收集57例急性淋巴細胞白血病患者的骨髓或外周血細胞,使用Ficoll梯度離心法分離單個核細胞,提取基因組DNA后采用PCR的方法對DNMT3A熱點突變所在的第23號外顯子進行擴增,PCR產(chǎn)物純化后測序,與正�；蚪M序列比對篩查突變結果。通過本科室生物標本庫獲取57例患者相關臨床特征信息并進行統(tǒng)計分析。 結果：在57例ALL患者中,共檢測出3例(5.3%)攜帶DNMT3A R882H突變。T-ALL患者中突變的發(fā)生率明顯高于B-ALL患者(P=0.048)。年齡在40至60歲之間的患者與其他年齡組相比具有更高的突變發(fā)生率(P=-0.042)。在全部患者中和T-ALL中,攜帶DNMT3A突變的患者與野生型患者相比整體生存期較短(P=0.02和P=0.014)。結論：DNMT3A突變在ALL中發(fā)生率較低,突變患者其整體生存期明顯縮短,提示該突變對成人ALL患者的危險分級和治療具有重要的意義。 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甲基轉移酶3A (DNA methyltransferase, DNMT3A)突變發(fā)生的頻率、與其他臨床特征間的相關性以及對患者預后的影響。方法：收集392例成人急性髓系白血病患者的骨髓或外周血細胞,使用Ficoll梯度離心法從中獲取單個核細胞,提取基因組DNA后采用PCR的方法對DNMT3A熱點突變所在的第23號外顯子進行擴增,PCR產(chǎn)物純化之后測序,最后與正�；蚪M序列比對判讀突變結果。通過本科室生物標本庫獲取392例患者相關臨床特征信息并進行統(tǒng)計分析。 結果：在392例AML患者中,共檢測出37例(9.44%)攜帶DNMT3A R882突變,其中DNMT3A R882H29例(78.38%),R882C8例(21.62%)。DNMT3A突變陽性患者與野生型患者相比,年齡明顯偏大,但在性別、白細胞計數(shù)、血紅蛋白水平、血小板計數(shù)和初診時原始細胞比例等方面無明顯差異。DNMT3A突變主要集中于M4和M5中,與NPM1突變和FLT3-ITD突變之間存在明顯正相關性(P0.001)。在染色體核型分析中,DNMT3A突變主要集中在CN-AML中(83.78%,31/37,P0.001)以及中危組中(91.89%,34/37,P0.001)。生存分析顯示,在整體AML和CN-AML患者中,DNMT3A突變陽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)均明顯縮短。在CN-AML中,多因素分析結果顯示DNMT3A突變可以作為一個獨立的預后因素,提示較短的整體生存期(HR=1.986,95%CI1.207to3.269,P=0.007)。 結論：DNMT3A突變在AML中發(fā)生頻率較高,并且對患者預后具有不良影響,提示該突變對成人AML患者預后危險分級和治療方案的選擇具有重要的意義。 目的：我們研究的目的在于利用TALENs技術構建一株攜帶DNMT3A突變的K562細胞系,并研究該突變對腫瘤細胞生物學效應的影響。 方法：設計并使用快速模塊組裝法構建DNMT3A-TALEN質(zhì)粒。TALEN質(zhì)粒和供者質(zhì)粒一起電轉入K562細胞,之后用PCR的方法篩選攜帶DNMT3A R882H突變的克隆。用全外顯子測序的方法檢測DNMT3A-TALEN質(zhì)�？赡艽嬖诘拿摪行�。蛋白質(zhì)印跡技術檢測DNMT3A和SLC7A11基因蛋白水平的表達變化�？寺⌒纬稍囼灪虲FSE實驗檢測突變克隆的增殖能力。用流式細胞術檢測細胞凋亡率。 結果：本研究中,我們首次使用TALENs技術成功構建了一株攜帶DNMT3A R882H突變的K562細胞系�？寺⌒纬稍囼灪虲FSE實驗證實該突變可以明顯促進細胞的增殖。表達譜芯片結果顯示一些與GSH合成相關的關鍵基因表達明顯升高,包括CTH,PSPH, PSAT1特別是SLC7A11(胱氨酸/谷氨酸轉運體),從而導致突變克隆細胞內(nèi)GSH含量升高。此外,檢測細胞凋亡率結果顯示突變克隆出現(xiàn)化療抵抗,聯(lián)合使用SSZ(柳氮磺胺吡啶)可以提高對化療藥物的敏感性。 結論：本研究結果為DNMT3A R882H突變在AML發(fā)病機制中的作用提供了新的見解,DNMT3A R882H突變克隆細胞內(nèi)GSH含量升高,出現(xiàn)化療抵抗,聯(lián)合使用SLC7A11的抑制劑SSZ可提高化療藥物敏感性,為攜帶DNMT3A R882H突變AML患者提供了新的診療思路。
[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.
【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R733.71

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