BRD4抑制劑JQ1對(duì)Jurkat細(xì)胞增殖抑制及凋亡作用的研究
發(fā)布時(shí)間：2018-09-05 08:54
【摘要】：目的:急性T淋巴細(xì)胞白血病(acute T lymphocytic leukemia,T-ALL)是來(lái)源于T淋巴母細(xì)胞的血液系統(tǒng)惡性克隆性疾病,因化療療效差、常規(guī)治療易復(fù)發(fā),同時(shí)易發(fā)生中樞神經(jīng)系統(tǒng)白血病成為臨床治療的難題,國(guó)內(nèi)外近期研究發(fā)現(xiàn),在T-ALL患者中,表現(xiàn)出明顯的c-myc基因成癮性,在抑制c-myc水平后,可以有效地緩解腫瘤進(jìn)展。近年來(lái)對(duì)溴結(jié)構(gòu)域蛋白(BRD)的研究發(fā)現(xiàn),其抑制劑(JQ1)可以明顯抑制c-myc基因,對(duì)多種腫瘤有抑制及殺傷作用,包括有慢性髓系白血病、卵巢癌、惡性膠質(zhì)瘤、肺腺癌等。本課題組在前期的研究中也發(fā)現(xiàn)JQ1可以明顯降低腫瘤細(xì)胞中c-myc基因的表達(dá)水平,從而對(duì)BCR-ABL基因陽(yáng)性的急性B淋巴細(xì)胞白血病株有明顯的治療效果。因此,JQ1對(duì)來(lái)源于胸腺祖細(xì)胞的T系急性淋巴細(xì)胞白血病,是否具有同樣的抑制作用,它的機(jī)制又是如何是本研究重點(diǎn)。本研究旨通過(guò)JQ1干預(yù)體外培養(yǎng)急性T淋巴細(xì)胞白血病細(xì)胞株(Jurkat細(xì)胞株),觀察細(xì)胞增殖抑制、凋亡作用,同時(shí)檢測(cè)NOTCH通路上Notch1、c-myc、PTEN基因表達(dá)情況,探討JQ1可能的抗T-ALL可能機(jī)制,為T(mén)-ALL的治療提供一條新的思路,并為臨床應(yīng)用提供理論基礎(chǔ)。方法:1四甲基偶氮唑藍(lán)(MTT)法檢測(cè)JQ1作用于Jurkat細(xì)胞株的增殖抑制率;2流式細(xì)胞術(shù)檢測(cè)Jurkat細(xì)胞的凋亡率改變;3實(shí)時(shí)熒光定量聚合酶鏈反應(yīng)(RT-PCR)法檢測(cè)Notch1、c-myc、PTEN mRNA的表達(dá)。結(jié)果:1.MTT檢測(cè)結(jié)果示:Jurkat細(xì)胞株和不同濃度的JQ1共同培養(yǎng)48h、72h、96h后,不同濃度JQ1對(duì)Jurkat細(xì)胞株增殖有明顯的抑制作用,且實(shí)驗(yàn)組的細(xì)胞增殖抑制率隨藥物濃度增加、作用時(shí)間延長(zhǎng)而升高,呈時(shí)間-劑量依賴性。JQ1在48 h的半數(shù)抑制濃度(IC50)為2.67μmol/L。2.流式細(xì)胞術(shù)檢測(cè)結(jié)果示:不同濃度JQ1干預(yù)Jurkat細(xì)胞48h、72h后,經(jīng)熒光染色流式細(xì)胞儀檢測(cè),結(jié)果顯示48 h時(shí)間點(diǎn)對(duì)照組早期凋亡率為(1.41±0.06)%,而觀察組JQ1在0.8、1.6、4μmol/L濃度時(shí)早期凋亡率分別為(20.9±0.67)%、(25.27±0.32)%、(32.70±0.93)%;而72 h對(duì)照組早期凋亡率為(16.50±0.46)%,實(shí)驗(yàn)組JQ1不同濃度組分別為(26.43±0.72)%,(39.67±0.64)%,(61.23±1.15)%。通過(guò)統(tǒng)計(jì)學(xué)單因素方差分析,同一時(shí)間點(diǎn)各濃度組有顯著差異,相同濃度組細(xì)胞凋亡率隨時(shí)間延長(zhǎng)明顯增加,與對(duì)照組相比較有統(tǒng)計(jì)學(xué)意義,表現(xiàn)為時(shí)間-劑量依賴性(時(shí)間效應(yīng)線性趨勢(shì)的F=373.845,P0.001,劑量線性趨勢(shì)的F=505.763,P0.001)。3.實(shí)時(shí)熒光定量PCR檢測(cè)結(jié)果示:不同藥物濃度處理Jurkat細(xì)胞48h后,RT-PCR檢測(cè)Notch1、c-Myc和PTEN相關(guān)基因的溶解曲線呈單一擴(kuò)增曲線,特異性較好。結(jié)果顯示各觀察組Notch1、c-myc基因表達(dá)量較對(duì)照組顯著下降,而PTEN mRNA表達(dá)量明顯增加,且隨藥物濃度增加基因表達(dá)量變化趨勢(shì)明顯(F值分別為1624.83、823.82、4460.76,P值均0.001),組間有顯著統(tǒng)計(jì)學(xué)差異(P0.05)。結(jié)論:1.JQ1可以明顯抑制Jurkat細(xì)胞增殖,并呈時(shí)間及劑量依賴性;2.JQ1可以有效誘導(dǎo)Jurkat細(xì)胞的凋亡,其凋亡效應(yīng)也呈時(shí)間及劑量依賴性;3.JQ1可以明顯抑制Jurkat細(xì)胞中NOTCH通道相關(guān)基因:Notch1基因、c-myc基因,同時(shí)上調(diào)PTEN基因的表達(dá),提示JQ1作用于Jurkat細(xì)胞靶點(diǎn)之一可能是通過(guò)NOTCH信號(hào)通路發(fā)揮作用。
[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.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：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.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R733.71
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