BRD4抑制剂JQ1对Jurkat细胞增殖抑制及凋亡作用的研究
发布时间：2018-09-05 08:54
【摘要】：目的:急性T淋巴细胞白血病(acute T lymphocytic leukemia,T-ALL)是来源于T淋巴母细胞的血液系统恶性克隆性疾病,因化疗疗效差、常规治疗易复发,同时易发生中枢神经系统白血病成为临床治疗的难题,国内外近期研究发现,在T-ALL患者中,表现出明显的c-myc基因成瘾性,在抑制c-myc水平后,可以有效地缓解肿瘤进展。近年来对溴结构域蛋白(BRD)的研究发现,其抑制剂(JQ1)可以明显抑制c-myc基因,对多种肿瘤有抑制及杀伤作用,包括有慢性髓系白血病、卵巢癌、恶性胶质瘤、肺腺癌等。本课题组在前期的研究中也发现JQ1可以明显降低肿瘤细胞中c-myc基因的表达水平,从而对BCR-ABL基因阳性的急性B淋巴细胞白血病株有明显的治疗效果。因此,JQ1对来源于胸腺祖细胞的T系急性淋巴细胞白血病,是否具有同样的抑制作用,它的机制又是如何是本研究重点。本研究旨通过JQ1干预体外培养急性T淋巴细胞白血病细胞株(Jurkat细胞株),观察细胞增殖抑制、凋亡作用,同时检测NOTCH通路上Notch1、c-myc、PTEN基因表达情况,探讨JQ1可能的抗T-ALL可能机制,为T-ALL的治疗提供一条新的思路,并为临床应用提供理论基础。方法:1四甲基偶氮唑蓝(MTT)法检测JQ1作用于Jurkat细胞株的增殖抑制率;2流式细胞术检测Jurkat细胞的凋亡率改变;3实时荧光定量聚合酶链反应(RT-PCR)法检测Notch1、c-myc、PTEN mRNA的表达。结果:1.MTT检测结果示:Jurkat细胞株和不同浓度的JQ1共同培养48h、72h、96h后,不同浓度JQ1对Jurkat细胞株增殖有明显的抑制作用,且实验组的细胞增殖抑制率随药物浓度增加、作用时间延长而升高,呈时间-剂量依赖性。JQ1在48 h的半数抑制浓度(IC50)为2.67μmol/L。2.流式细胞术检测结果示:不同浓度JQ1干预Jurkat细胞48h、72h后,经荧光染色流式细胞仪检测,结果显示48 h时间点对照组早期凋亡率为(1.41±0.06)%,而观察组JQ1在0.8、1.6、4μmol/L浓度时早期凋亡率分别为(20.9±0.67)%、(25.27±0.32)%、(32.70±0.93)%;而72 h对照组早期凋亡率为(16.50±0.46)%,实验组JQ1不同浓度组分别为(26.43±0.72)%,(39.67±0.64)%,(61.23±1.15)%。通过统计学单因素方差分析,同一时间点各浓度组有显著差异,相同浓度组细胞凋亡率随时间延长明显增加,与对照组相比较有统计学意义,表现为时间-剂量依赖性(时间效应线性趋势的F=373.845,P0.001,剂量线性趋势的F=505.763,P0.001)。3.实时荧光定量PCR检测结果示:不同药物浓度处理Jurkat细胞48h后,RT-PCR检测Notch1、c-Myc和PTEN相关基因的溶解曲线呈单一扩增曲线,特异性较好。结果显示各观察组Notch1、c-myc基因表达量较对照组显著下降,而PTEN mRNA表达量明显增加,且随药物浓度增加基因表达量变化趋势明显(F值分别为1624.83、823.82、4460.76,P值均0.001),组间有显著统计学差异(P0.05)。结论:1.JQ1可以明显抑制Jurkat细胞增殖,并呈时间及剂量依赖性;2.JQ1可以有效诱导Jurkat细胞的凋亡,其凋亡效应也呈时间及剂量依赖性;3.JQ1可以明显抑制Jurkat细胞中NOTCH通道相关基因:Notch1基因、c-myc基因,同时上调PTEN基因的表达,提示JQ1作用于Jurkat细胞靶点之一可能是通过NOTCH信号通路发挥作用。
[Abstract]:Objective: Acute T lymphocytic leukemia (T-ALL) is a malignant clonal disease of the blood system originated from T lymphoblasts. Because of the poor efficacy of chemotherapy, routine treatment is easy to recur, and the occurrence of central nervous system leukemia becomes a difficult problem in clinical treatment. Recent studies at home and abroad have found that in T-ALL patients, the performance of the disease. Recent studies on brominated domain protein (BRD) have shown that its inhibitor (JQ1) can significantly inhibit the c-myc gene and inhibit and kill many tumors, including chronic myeloid leukemia, ovarian cancer, malignant glioma and lung adenocarcinoma. Our group also found that JQ1 can significantly reduce the expression of c-myc gene in tumor cells, thus has a significant therapeutic effect on BCR-ABL gene positive acute B-lymphocytic leukemia. Therefore, whether JQ1 has the same inhibitory effect on T-line acute lymphocytic leukemia derived from thymic progenitor cells? The aim of this study is to investigate the possible mechanism of JQ1 against T-ALL by interfering with the proliferation inhibition and apoptosis of acute T-lymphoblastic leukemia cell line (Jurkat cell line) in vitro and detecting the expression of Notch1, c-myc and PTEN genes in NOTCH pathway. Methods: 1. MTT assay was used to detect the inhibitory rate of JQ1 on Jurkat cell lines, 2 flow cytometry was used to detect the apoptosis rate of Jurkat cells, 3 real-time fluorescence quantitative polymerase chain reaction (RT-PCR) was used to detect the expression of Notch1, c-myc, PTEN mRNA. The results showed that the proliferation of Jurkat cell line was inhibited by different concentrations of JQ1 after 48 hours, 72 hours and 96 hours of co-culture. The inhibition rate of JQ1 on Jurkat cell proliferation increased in a time-dose dependent manner with the increase of drug concentration and the prolongation of action time. The half inhibitory concentration (IC50) of JQ1 at 48 hours was 2.67 micron. Ol/L.2. Flow cytometry showed that the early apoptosis rate of Jurkat cells was (1.41.06)% in the control group at 48 h after intervention with different concentrations of JQ1 for 48 h, and (20.9.67)% (25.27.32)% (32.70.93)% in the observation group at 0.8, 1.6 and 465507 The early apoptotic rate was (16.50.46)% in the control group at 72 h, and (26.43.72)%, (39.67.64)%, (61.23.15)% in the experimental group at different concentrations of JQ1, respectively. Significance: Time-dose dependence (F = 373.845, P 0.001, F = 505.763, P 0.001, F = 505.763, P 0.001). 3. Real-time fluorescence quantitative PCR assay showed that after 48 hours of treatment with different drug concentrations, Notch1, c-Myc and PTEN-related genes were detected by RT-PCR with a single amplification curve. The results showed that the expression of Notch1 and c-myc genes in each observation group was significantly lower than that in the control group, while the expression of PTEN mRNA was significantly increased, and the expression of PTEN mRNA was significantly changed with the increase of drug concentration (F values were 1624.83, 823.82, 4460.76, P values were 0.001, respectively). There was a significant difference between the two groups (P 0.05). Conclusion: 1. JQ1 can significantly inhibit Jurkat cells. JQ1 can effectively induce apoptosis of Jurkat cells in a time-and dose-dependent manner; 3. JQ1 can significantly inhibit NOTCH channel-related genes in Jurkat cells: Notch1 gene, c-myc gene, and up-regulate the expression of PTEN gene, suggesting that JQ1 may be one of the targets of Jurkat cells. It plays a role through the NOTCH signaling pathway.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R733.71
[Abstract]:Objective: Acute T lymphocytic leukemia (T-ALL) is a malignant clonal disease of the blood system originated from T lymphoblasts. Because of the poor efficacy of chemotherapy, routine treatment is easy to recur, and the occurrence of central nervous system leukemia becomes a difficult problem in clinical treatment. Recent studies at home and abroad have found that in T-ALL patients, the performance of the disease. Recent studies on brominated domain protein (BRD) have shown that its inhibitor (JQ1) can significantly inhibit the c-myc gene and inhibit and kill many tumors, including chronic myeloid leukemia, ovarian cancer, malignant glioma and lung adenocarcinoma. Our group also found that JQ1 can significantly reduce the expression of c-myc gene in tumor cells, thus has a significant therapeutic effect on BCR-ABL gene positive acute B-lymphocytic leukemia. Therefore, whether JQ1 has the same inhibitory effect on T-line acute lymphocytic leukemia derived from thymic progenitor cells? The aim of this study is to investigate the possible mechanism of JQ1 against T-ALL by interfering with the proliferation inhibition and apoptosis of acute T-lymphoblastic leukemia cell line (Jurkat cell line) in vitro and detecting the expression of Notch1, c-myc and PTEN genes in NOTCH pathway. Methods: 1. MTT assay was used to detect the inhibitory rate of JQ1 on Jurkat cell lines, 2 flow cytometry was used to detect the apoptosis rate of Jurkat cells, 3 real-time fluorescence quantitative polymerase chain reaction (RT-PCR) was used to detect the expression of Notch1, c-myc, PTEN mRNA. The results showed that the proliferation of Jurkat cell line was inhibited by different concentrations of JQ1 after 48 hours, 72 hours and 96 hours of co-culture. The inhibition rate of JQ1 on Jurkat cell proliferation increased in a time-dose dependent manner with the increase of drug concentration and the prolongation of action time. The half inhibitory concentration (IC50) of JQ1 at 48 hours was 2.67 micron. Ol/L.2. Flow cytometry showed that the early apoptosis rate of Jurkat cells was (1.41.06)% in the control group at 48 h after intervention with different concentrations of JQ1 for 48 h, and (20.9.67)% (25.27.32)% (32.70.93)% in the observation group at 0.8, 1.6 and 465507 The early apoptotic rate was (16.50.46)% in the control group at 72 h, and (26.43.72)%, (39.67.64)%, (61.23.15)% in the experimental group at different concentrations of JQ1, respectively. Significance: Time-dose dependence (F = 373.845, P 0.001, F = 505.763, P 0.001, F = 505.763, P 0.001). 3. Real-time fluorescence quantitative PCR assay showed that after 48 hours of treatment with different drug concentrations, Notch1, c-Myc and PTEN-related genes were detected by RT-PCR with a single amplification curve. The results showed that the expression of Notch1 and c-myc genes in each observation group was significantly lower than that in the control group, while the expression of PTEN mRNA was significantly increased, and the expression of PTEN mRNA was significantly changed with the increase of drug concentration (F values were 1624.83, 823.82, 4460.76, P values were 0.001, respectively). There was a significant difference between the two groups (P 0.05). Conclusion: 1. JQ1 can significantly inhibit Jurkat cells. JQ1 can effectively induce apoptosis of Jurkat cells in a time-and dose-dependent manner; 3. JQ1 can significantly inhibit NOTCH channel-related genes in Jurkat cells: Notch1 gene, c-myc gene, and up-regulate the expression of PTEN gene, suggesting that JQ1 may be one of the targets of Jurkat cells. It plays a role through the NOTCH signaling pathway.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R733.71
【相似文献】
相关期刊论文 前10条
1 韩燕;王淑红;孟照俊;张富军;宁启兰;;热处理对HeLa细胞增殖、凋亡及HSP70表达的影响[J];陕西医学杂志;2011年04期
2 卜仁戈,吴斌,宋永胜,费翔;Genistein对EJ细胞增殖抑制作用的研究[J];中国医科大学学报;2005年04期
3 钟东明;蔡静怡;肖云;李庆华;朱如芳;;苯对细胞增殖抑制率的影响及保护因素探讨[J];中国热带医学;2013年07期
4 王婷婷;刘继文;王生玲;张亚楼;;磷制剂对砷染毒细胞增殖抑制作用影响的实验研究[J];地方病通报;2007年03期
5 陈晓伟;王晓宁;龚，
本文编号：2223800
本文链接：https://www.wllwen.com/yixuelunwen/zlx/2223800.html
