本文關(guān)鍵詞： 雷公藤甲素 MV411細(xì)胞 PI3K-AKT-mTOR通路 PTEN　出處：《山西醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:急性髓系白血病(acute myelocytic leukemia,AML)是起源于骨髓造血干、祖細(xì)胞的惡性克隆性疾病。FLT3/ITD突變是AML中重要的基因突變之一,也是提示疾病預(yù)后不良的重要標(biāo)志。該類患者常規(guī)化療療效差,目前AML伴FLT3/ITD突變患者靶向藥物治療成為人們的研究熱點(diǎn),并且在臨床應(yīng)用上取得一定的療效。然而已有的靶向治療的耐藥和緩解后再復(fù)發(fā)成為AML伴FLT3/ITD突變病人治療的一大難題,因此尋求新的治療藥物及新的作用靶點(diǎn)十分必要。近年來中藥雷公藤因其抗炎、免疫抑制、抗腫瘤等多種藥理作用,備受國內(nèi)外學(xué)者關(guān)注。雷公藤甲素(TP)是雷公藤主要活性成分之一,相關(guān)文獻(xiàn)研究報道顯示,TP不論對急性髓系白血病細(xì)胞株HL60還是對慢性髓系白血病K562、K562G01細(xì)胞均有明顯增殖抑制作用。那么TP對FLT3/ITD突變的MV411細(xì)胞是否也有增殖抑制作用?其可能作用機(jī)制又是如何?本課題通過體外實(shí)驗(yàn)的方法觀察TP對MV411細(xì)胞的增殖抑制、凋亡作用及PI3K-AKT-mTOR通路相關(guān)基因FLT3、PTEN、PI3K、AKT、mTOR表達(dá),初步探討其可能的抗腫瘤機(jī)制,為AML伴FLT3/ITD突變患者的治療提供理論依據(jù)。研究方法:1四甲基偶氮唑藍(lán)實(shí)驗(yàn)(MTT)測定不同濃度的TP在24h,48h,72h對MV411細(xì)胞株的增殖抑制情況,并測算IC50值;2流式細(xì)胞儀測定48h,72h的細(xì)胞凋亡率;3實(shí)時熒光定量PCR檢測PI3K-AKT-mTOR通路相關(guān)基因FLT3,PTEN,PI3K,AKT,mTOR的表達(dá)水平。研究結(jié)果:1.MTT檢測結(jié)果示:TP對MV411細(xì)胞有明顯增殖抑制作用,且隨著藥物濃度和用藥時間的增加而逐漸增強(qiáng),呈現(xiàn)時間-劑量依賴性,選擇48小時為TP的IC50,值為16.92nmol/L。2.流式細(xì)胞儀檢測結(jié)果示:48小時時間點(diǎn)0、10、20nmol/L TP對應(yīng)的平均早期凋亡率分別為(3.30±0.20)%、(17.10±0.36)%、(35.67±0.61)%;72小時時間點(diǎn)0、10、20nmol/L TP對應(yīng)的平均早期凋亡率分別為(7.37±0.32)%、(49.33±0.40)%、(68.92±0.11)%。發(fā)現(xiàn)細(xì)胞凋亡率隨著濃度的增加而增加。3.實(shí)時熒光定量PCR檢測結(jié)果示:選用0、5、10、20nmol/L的TP作用于MV411細(xì)胞48h后,運(yùn)用實(shí)時熒光定量PCR法檢測,發(fā)現(xiàn)FLT3、PI3K、AKT、mTOR的m RNA水平下降,PTEN的m RNA水平升高,且呈現(xiàn)出劑量依賴性。結(jié)論:1.TP對MV411細(xì)胞有明顯增殖抑制作用,呈現(xiàn)時間-劑量依賴性;2.TP可以誘導(dǎo)MV411細(xì)胞凋亡;3.TP作用于MV411細(xì)胞,FLT3、PI3K、AKT、mTOR的m RNA水平下降,PTEN的m RNA水平升高。TP可能是通過影響PI3K-AKT-mTOR通路上相關(guān)基因表達(dá),誘導(dǎo)腫瘤細(xì)胞凋亡。
[Abstract]:Objective: acute myelocytic leukemia (AMLs) is derived from bone marrow hematopoietic stem. FLT3 / ITD mutation of progenitor cells is one of the important gene mutations in AML. It is also an important indicator of poor prognosis of the disease. The efficacy of conventional chemotherapy is poor in this kind of patients. At present, targeted drug therapy in patients with AML with FLT3/ITD mutation has become a hot research topic. However, drug resistance and relapse after remission have become a major problem in the treatment of AML patients with FLT3/ITD mutation. Therefore, it is very necessary to seek new therapeutic drugs and new targets. In recent years, Tripterygium wilfordii, due to its anti-inflammatory, immunosuppressive, anti-tumor and other pharmacological effects, Thetripterygium wilfordii (TPN) is one of the main active components of Tripterygium wilfordii. It has been reported that TP can inhibit the proliferation of both HL60 cells and K562K562G01 cells. Does TP inhibit the proliferation of MV411 cells with FLT3/ITD mutation? What is the possible mechanism? The aim of this study was to investigate the inhibitory effect of TP on the proliferation and apoptosis of MV411 cells and the expression of PI3K-AKT-mTOR pathway related gene (FLT3PTENN) PI3KPI3KPK (AKTT) mTOR in vitro, and to explore its possible anti-tumor mechanism. To provide a theoretical basis for the treatment of AML patients with FLT3/ITD mutation. Methods the proliferation inhibition of different concentrations of TP on MV411 cell lines at 24 h and 48 h for 72 h was determined by using 1: 1 tetramethyl azolium blue assay. The apoptosis rate was measured by flow cytometry for 48 h and 72 h. The expression level of PI3K-AKT-mTOR pathway related gene FLT3PTENTENTEN PI3KTK TOR was detected by real-time fluorescence quantitative PCR. The results showed that: 1. The cell proliferation of MV411 cells was significantly inhibited by the presence of 1: 1. The results showed that: TP could inhibit the proliferation of MV411 cells. And with the increase of drug concentration and time, it gradually increased, showing a time-dose dependent, The IC50 with 48 hours TP was 16.92nmol / L 路L ~ (-2). Flow cytometry showed that the average early apoptotic rate was 3.30 鹵0.20 鹵17.10 鹵0.36 鹵35.67 鹵0.612 nmol 路L ~ (-1) L ~ (TP) at 010 ~ (10) nmol / L ~ (-1) L ~ (-1) 路min ~ (-1) 路min ~ (-1) 路L ~ (-1), respectively. The average early apoptosis rate was 7.37 鹵0.32 鹵0.32 鹵0.40 nmol / L TP, respectively. It was found that the average early apoptotic rate was 7.37 鹵0.32 鹵0.40 nmol / L TP, respectively. The average early apoptosis rate was 7.37 鹵0.32 鹵0.40 nmol / L TP, respectively. The rate of death increased with the increase of concentration. The results of real-time fluorescence quantitative PCR showed that the MV411 cells were treated with 10 nmol / L TP for 48 h. Using real-time fluorescence quantitative PCR assay, it was found that the level of m RNA of FLT3 + PI3KAK TOR decreased and the level of m RNA of PTEN increased in a dose-dependent manner. Conclusion: 1. TP has a significant inhibitory effect on the proliferation of MV411 cells. In a time-dose dependent manner, TP could induce apoptosis of MV411 cells. 3. TP could induce apoptosis of MV411 cells by decreasing the level of m RNA of MV411 cells. TP may affect the expression of related genes in PI3K-AKT-mTOR pathway and induce apoptosis of tumor cells.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R733.71

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