雷公藤甲素对MV411细胞增殖抑制及在PI3K-AKT-mTOR通路机制研究

发布时间：2018-03-01 05:20

本文关键词： 雷公藤甲素 MV411细胞 PI3K-AKT-mTOR通路 PTEN　出处：《山西医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:急性髓系白血病(acute myelocytic leukemia,AML)是起源于骨髓造血干、祖细胞的恶性克隆性疾病。FLT3/ITD突变是AML中重要的基因突变之一,也是提示疾病预后不良的重要标志。该类患者常规化疗疗效差,目前AML伴FLT3/ITD突变患者靶向药物治疗成为人们的研究热点,并且在临床应用上取得一定的疗效。然而已有的靶向治疗的耐药和缓解后再复发成为AML伴FLT3/ITD突变病人治疗的一大难题,因此寻求新的治疗药物及新的作用靶点十分必要。近年来中药雷公藤因其抗炎、免疫抑制、抗肿瘤等多种药理作用,备受国内外学者关注。雷公藤甲素(TP)是雷公藤主要活性成分之一,相关文献研究报道显示,TP不论对急性髓系白血病细胞株HL60还是对慢性髓系白血病K562、K562G01细胞均有明显增殖抑制作用。那么TP对FLT3/ITD突变的MV411细胞是否也有增殖抑制作用?其可能作用机制又是如何?本课题通过体外实验的方法观察TP对MV411细胞的增殖抑制、凋亡作用及PI3K-AKT-mTOR通路相关基因FLT3、PTEN、PI3K、AKT、mTOR表达,初步探讨其可能的抗肿瘤机制,为AML伴FLT3/ITD突变患者的治疗提供理论依据。研究方法:1四甲基偶氮唑蓝实验(MTT)测定不同浓度的TP在24h,48h,72h对MV411细胞株的增殖抑制情况,并测算IC50值;2流式细胞仪测定48h,72h的细胞凋亡率;3实时荧光定量PCR检测PI3K-AKT-mTOR通路相关基因FLT3,PTEN,PI3K,AKT,mTOR的表达水平。研究结果:1.MTT检测结果示:TP对MV411细胞有明显增殖抑制作用,且随着药物浓度和用药时间的增加而逐渐增强,呈现时间-剂量依赖性,选择48小时为TP的IC50,值为16.92nmol/L。2.流式细胞仪检测结果示:48小时时间点0、10、20nmol/L TP对应的平均早期凋亡率分别为(3.30±0.20)%、(17.10±0.36)%、(35.67±0.61)%;72小时时间点0、10、20nmol/L TP对应的平均早期凋亡率分别为(7.37±0.32)%、(49.33±0.40)%、(68.92±0.11)%。发现细胞凋亡率随着浓度的增加而增加。3.实时荧光定量PCR检测结果示:选用0、5、10、20nmol/L的TP作用于MV411细胞48h后,运用实时荧光定量PCR法检测,发现FLT3、PI3K、AKT、mTOR的m RNA水平下降,PTEN的m RNA水平升高,且呈现出剂量依赖性。结论:1.TP对MV411细胞有明显增殖抑制作用,呈现时间-剂量依赖性;2.TP可以诱导MV411细胞凋亡;3.TP作用于MV411细胞,FLT3、PI3K、AKT、mTOR的m RNA水平下降,PTEN的m RNA水平升高。TP可能是通过影响PI3K-AKT-mTOR通路上相关基因表达,诱导肿瘤细胞凋亡。
[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 卤0.20 卤17.10 卤0.36 卤35.67 卤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 卤0.32 卤0.32 卤0.40 nmol / L TP, respectively. It was found that the average early apoptotic rate was 7.37 卤0.32 卤0.40 nmol / L TP, respectively. The average early apoptosis rate was 7.37 卤0.32 卤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.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.71

【参考文献】