急性T淋巴细胞白血病中STAT5B基因突变及靶向药物筛选

发布时间：2018-06-13 18:11

本文选题：STAT5B + T-ALL　；参考：《苏州大学》2015年硕士论文

【摘要】：目的1、分析阐述苏州大学附属第一医院急性T淋巴细胞白血病(T-ALL)的临床特点及实验室特征。2、探讨T-ALL患者及本中心保存的8种T-ALL细胞株STAT5B基因突变。3、筛选针对STAT5B基因突变的靶向药物,初步阐述其作用机理。研究对象和方法1、研究对象:收集2006年1月至2014年7月在苏州大学附属第一医院初诊急性T淋巴细胞白血病(T-ALL)的141例患者的临床及实验室资料。其中,男性116例,女性25例,男女性别比例为4.64:1;成年患者(18岁)93例,儿童患者("f18岁)48例,患者中位年龄为24岁。2、选取93例急性T淋巴细胞白血病患者及本中心保存的8种T-ALL细胞株,抽提RNA,并设计引物通过RT-PCR扩增STAT5B基因全长,所得RT-PCR产物采用第一代测序技术Sanger测序法分析STAT5B基因突变。3、以伴有STAT5B N642H位点基因突变的KOPT-K1细胞株为细胞模型,应用CCK8法分析初步筛选STAT5B N642H位点基因突变靶向药物。4、应用流式细胞术(flow cytometry,FCM)检测候选药物第三代TKI Ponatinib处理KOPT-K1细胞后的凋亡及周期变化。结果急性T淋巴细胞白血病患者临床及实验室特征1、141例急性T淋巴细胞白血病患者,白细胞计数中位值为93×109/L,血红蛋白含量中位值为105g/L,血小板计数中位值为60×109/L,骨髓原始细胞比例中位值为83.5%。按照FAB分型标准,141例T-ALL患者中ALL-L1型40例,ALL-L2型46例,ALL未分类型(NOS)55例。三组患者临床基本信息统计分析发现,在初诊骨髓原幼细胞比例中位值方面,ALL-L1ALL-L2NOS(88%84.5%76%,P0.05);在年龄、性别、中位白细胞计数、中位血红蛋白含量、中位血小板计数方面,三组之间无统计学差异。2、141例T-ALL患者中,98.6%(139/141)的患者染色体核型分析成功,染色体正常核型(NK)占58.3%(81/139),异常核型检出率为41.7%(58/139)。其中,单体核型(MK)占5.8%(8/139),t(11;14)(p13;q11)占5.0%(7/139),其它≥2次的结构性异常主要有6q-,del(6)(q21),i(7q),+8。3、83例T-ALL患者进行融合基因检测,20例患者可检测到基因异常。4例男性患者检测到SET-NUP214融合基因转录本,中位年龄为26(23-54)岁;8例患者检测到SIL-TAL1融合基因转录本,中位年龄为17.5(12-42)岁,男女性别比为7:1。其他检测到的融合基因有:SET-CAN(+)2例,BCR-ABL(+)1例,HOX11(+)1例,MLL-MLL(+)1例,MLL-AF10(+)1例,MLL-AF9(+)1例,TCR重排(+)1例。4、对117例T-ALL患者进行常见基因突变检测(PHF6、FBXW7、NOTCH1、WT1、LMO2),其中,NOTCH1突变最常见,发生率为47%(55/117),16例患者具有PHF6突变(13.7%),FBXW7、WT1、LMO2突变例数分别为12例(10.2%)、4例(3.4%)、3例(2.6%)。急性T淋巴细胞白血病患者及T-ALL细胞株中STAT5B基因突变研究Sanger测序结果显示93例T-ALL患者及8株T-ALL细胞株中只发现STAT5B N642H位点基因突变。其中,1例儿童患者伴有STAT5B p.N642H突变,8株T-ALL细胞株中KOPT-K1细胞株存在STAT5B N642H突变。STAT5B基因突变靶向药物筛选1、4种候选药物分析发现,酪氨酸激酶抑制剂(TKI)及STAT5B抑制剂AZD1480对KOPT-K1细胞均有抑制作用。其中,第3代TKI Ponatinib对其抑制作用最强,其抑制作用优于STAT5B抑制剂AZD1480。2、第3代TKI Ponatinib处理KOPT-K1细胞72h后,流式细胞周期检测发现G0/G1细胞比例明显高于未加药组(75.61±0.12%VS 58.88±4.07%,P0.05)。结论1、本中心141例急性T淋巴细胞白血病患者中,异常染色体及再现性染色体异常检出率较低(41.7%),最常见的融合基因为SIL-TAL1,NOTCH1突变在T-ALL中发生率最高。2、成人T-ALL患者中STAT5B N642H位点突变罕见,儿童中STAT5B N642H突变发生率为3.6%,发现KOPT-K1细胞系存在STAT5B N642H位点基因突变,为研究STAT5B基因突变的药物提供理想的细胞模型。3、第三代TKI Ponatinib对KOPT-K1细胞的增殖有抑制作用,且KOPT-K1细胞对Ponatinib最为敏感,提示小分子药物Ponatinib在治疗STAT5B基因突变的血液病中可能具有临床应用价值。
[Abstract]:Objective 1 to analyze the clinical characteristics and laboratory characteristics of acute T lymphocytic leukemia (T-ALL) in First Hospital Affiliated to Suzhou University,.2, to explore the STAT5B gene mutation.3 of T-ALL patients and the 8 T-ALL cell lines preserved in this center, and to screen the target drugs for the mutation of STAT5B gene. The research object and method 1, the research object and method, are discussed. Objective: to collect the clinical and laboratory data of 141 patients with acute T lymphocytic leukemia (T-ALL) in First Hospital Affiliated to Suzhou University from January 2006 to July 2014, including 116 male and 25 female, 25 male and female sex ratio 4.64:1, 93 adult patients (18 years old), 48 cases of children ("F18 years old"), and the median age of 24 years.2, 93 patients with acute T lymphocytic leukemia and 8 T-ALL cell lines preserved in this center were selected to extract RNA, and primers were designed to amplify the full length of the STAT5B gene by RT-PCR. The RT-PCR products were analyzed by the first generation sequencing technique and Sanger sequencing method to analyze the STAT5B gene mutation.3, and the KOPT-K1 cell line with the STAT5B N642H site gene mutation was fine. Cell model, CCK8 method was used to preliminarily screen STAT5B N642H loci gene mutation targeting drug.4, and the apoptosis and cycle changes after third generation TKI Ponatinib processing KOPT-K1 cells were detected by flow cytometry (flow cytometry, FCM). Results of acute T lymphocyte leukopathy, 1141 cases of acute T lymphatic lymph nodes were characterized by clinical and laboratory characteristics. In leukemic patients, the median value of leukocyte count was 93 x 109/L, the median value of hemoglobin content was 105g/L, the value of the platelet count was 60 x 109/L, the median of bone marrow cell proportion was 83.5%. according to the FAB classification standard, 141 cases of T-ALL patients were ALL-L1 type, 46 cases of ALL-L2, 55 cases of ALL undivided (NOS). Information statistical analysis found that ALL-L1ALL-L2NOS (88%84.5%76%, P0.05) in the proportion of primary myeloid cells in the first diagnosis; there was no statistical difference between the three groups in age, sex, median white blood cell count, median hemoglobin content, and median platelet count, and 98.6% (139/141) of the patients with T-ALL. The chromosomal normal karyotype (NK) was 58.3% (81/139), and the abnormal karyotype detection rate was 41.7% (58/139). Among them, the monosomal karyotype (MK) accounted for 5.8% (8/139), t (11; 14) (P13; Q11) accounted for 5% (7/139), and the other 2 Structural abnormalities were mainly 6q-, del (6) (q21), and 20 patients could detect gene differentiation. The SET-NUP214 fusion gene transcriptional transcript was detected in.4 male patients, and the median age was 26 (23-54) years; 8 patients detected the SIL-TAL1 fusion gene transcriptional transcript, the median age was 17.5 (12-42) years, and the sex ratio of men and women was 7:1. (2 cases, +) 1 cases, HOX11 (+) 1 cases, MLL-MLL (+) 1 cases, MLL-AF10 (+) 1 cases, M, M, M, M, M, and M. LL-AF9 (+) 1 cases, TCR rearrangement (+) 1 cases of.4, 117 cases of T-ALL patients with common gene mutation detection (PHF6, FBXW7, NOTCH1, WT1, LMO2), of which NOTCH1 mutation is the most common, the incidence is 47% (55/117), 16 cases with PHF6 mutations (10.2%), 4 cases (3.4%), 3 cases (2.6%). Acute lymphoblastic leukemia. The Sanger sequencing of STAT5B gene mutations in patients and T-ALL cells showed that only 93 cases of T-ALL and 8 T-ALL cell lines found only the mutation of STAT5B N642H loci, of which 1 children were accompanied by STAT5B p.N642H mutation, and the KOPT-K1 cell line of 8 T-ALL cell lines had a mutant targeting drug sieve. The selection of 1,4 candidate drug analysis showed that tyrosine kinase inhibitor (TKI) and STAT5B inhibitor AZD1480 had inhibitory effect on KOPT-K1 cells. Among them, the third generation TKI Ponatinib had the strongest inhibitory effect. The inhibitory effect was better than the STAT5B inhibitor AZD1480.2, and the third generation TKI Ponatinib treated KOPT-K1 cells. The proportion of /G1 cells was significantly higher than that in the untreated group (75.61 + 0.12%VS 58.88 + 4.07%, P0.05). Conclusion 1. Among 141 patients with acute T lymphocytic leukemia, abnormal chromosomes and reproducible chromosomal abnormalities were low (41.7%), the most common fusion gene was SIL-TAL1, NOTCH1 mutation was the highest in T-ALL and S in adult T-ALL patients. The mutation of TAT5B N642H site is rare, and the incidence of STAT5B N642H mutation in children is 3.6%. It is found that there is a STAT5B N642H locus mutation in the KOPT-K1 cell line, which provides an ideal cell model.3 for the study of the STAT5B gene mutation. The third generation TKI Ponatinib can inhibit the proliferation of the KOPT-K1 cells, and the cells are most sensitive to the STAT5B. It is suggested that small molecule Ponatinib may be of clinical value in the treatment of STAT5B gene mutation hematopathy.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R733.71

【相似文献】