双氢青蒿素联合顺铂诱导人肺腺癌H1299细胞凋亡及其机制研究

发布时间：2018-05-27 13:28

本文选题：双氢青蒿素 + 顺铂　；参考：《河北医科大学》2017年硕士论文

【摘要】：目的:肺癌的发病率及相关死亡率在目前全球癌症中所占比例最高。其中80%的肺癌病人为非小细胞肺癌(NSCLC)。目前临床一线使用的含铂化疗方案有效率仅有30%。因此,大家迫切的需要找到一种新的化疗药物来提高肺癌病人的治疗效率。近年的许多研究中,青蒿素及其衍生物在一些对其他药物敏感及耐药的肿瘤细胞株中均展现出了强大的抗癌活性。双氢青蒿素(DHA)—一种已知的青蒿素的衍生物,这些年来,它的抗癌活性在世界范围内被各国学者广泛研究。而双氢青蒿素也展现出了对肿瘤细胞株如神经胶质瘤细胞、乳腺癌细胞、结肠癌细胞、肺癌细胞、卵巢癌细胞、胰腺癌细胞的抗癌作用。大量的研究表明许多肿瘤细胞p53基因的缺失或未表达均与化疗的耐药有关。但迄今为止,尚未查到关于双氢青蒿素联合顺铂(Cis)作用于p53缺失型人肺腺癌H1299细胞系的相关研究。而这项实验的设计初衷就是研究双氢青蒿素联合顺铂诱导H1299细胞凋亡的协同作用及其潜在的作用机制。方法:1体外培养人类肺腺癌H1299细胞并将细胞分为四组:双氢青蒿素(DHA)组、顺铂(Cis)组、联合用药组及对照组,通过四甲基偶氮唑盐/噻唑蓝(MTT)实验来评价不同处理组间H1299细胞抑制率的变化情况。2采用p53基因正常表达的A549细胞作为对照,通过MTT实验比较在相同药物处理条件下,DHA及Cis分别对A549细胞和H1299细胞抑制率的影响。3采用流式细胞术(FCM)来评价DHA作用不同时间、剂量的情况下,对H1299细胞的凋亡率的影响。4通过赫斯特33258(Hoechst 33258)对细胞核染色来观察经DHA处理后H1299细胞核的形态变化。5通过蛋白印迹法(Western Blot)和逆转录实时定量聚合酶链反应(RT-q PCR)来评价凋亡相关通路蛋白及基因的表达情况。结果:1 MTT实验和Hoechst 33258细胞核染色实验显示随着各自药物浓度的增加以及作用时间的延长双氢青蒿素和顺铂单药处理组细胞凋亡率明显增加,联合用药处理组的细胞凋亡率更高。另一个MTT实验结果显示:相同浓度的Cis孵育A549及H1299两组细胞48小时后,对A549细胞的抑制率明显高于对H1299细胞的抑制率。于此相反,使用相同浓度的DHA孵育上述两组细胞48小时后,两组间的细胞抑制率均无明显差异;2流式细胞术的结果显示当DHA浓度为20μM时,H1299细胞的凋亡率从9.09%(对照组)上升至17.07%(24小时)以及24.31%(48小时);当DHA作用于相同时间(24小时),其引起细胞的凋亡率从9.09%(对照组)上升至17.07%(浓度为20μM)以及22.5%(浓度为40μM);3 RT-q PCR的结果显示单药DHA(20μM)、Cis(12.5μM)及两药联合孵育24小时及48小时,H1299细胞中半胱天冬氨酸蛋白酶(Caspase)-3、-8、-9m RNA的表达均上调,其中联合用药组Caspase-3,-8,-9的2-(35)(35)Ct值高于单药组,而单药组Caspase3,8,9的2-(35)(35)Ct值高于对照组;4 Western Blot结果显示联合用药组能显著提高Caspase-8,-9,-3,-6,-7和多腺苷二磷酸核糖聚合酶(PARP)的活性。提前在H1299细胞中加入Caspase-3、-8、-9抑制剂后,再次通过Western Blot实验观察这三个蛋白的表达情况,结果表明提前加入z DQMD-fmk组的细胞,Cleavecaspase-3的表达明显降低,但是Cleave-caspase-8、-9的表达无明显变化。而提前加入z IETD-fmk组的细胞,显著降低了Cleave-caspase-8的表达,部分降低了Cleave-caspase-3、-9的表达。提前孵育z LEHD-fmk组的细胞Cleave-caspase-9的表达显著降低,Cleave-caspase-3的表达部分降低,而Cleave-caspase-8的表达基本无变化。结论:1 DHA作用于H1299细胞能诱导时间及剂量依赖性细胞毒性。2 DHA作用于H1299细胞能诱导时间及剂量依赖性的细胞凋亡。3 DHA联合Cis作用于H1299细胞有协同促凋亡作用。4 Caspase-8参与的死亡受体信号通路及Caspase-9参与的线粒体信号通路与DHA及CIS联合用药产生的协同作用有关。5 DHA及Cis单药及联合用药均能上调H1299细胞Caspase-3,-8,-9m RNA的表达。综上所述,本研究结果表明双氢青蒿素联合顺铂可能通过Caspase-8参与的死亡受体凋亡通路和Caspase-9参与的线粒体凋亡通路产生协同作用诱导H1299细胞凋亡。
[Abstract]:Objective: the incidence and mortality of lung cancer are the highest in the current global cancer. 80% of the patients with lung cancer are non small cell lung cancer (NSCLC). At present, the effective rate of platinum chemotherapy is only 30%., so it is urgent to find a new chemotherapeutic agent to improve the efficiency of lung cancer patients. In many studies in recent years, artemisinin and its derivatives have shown strong anti-cancer activity in some cancer cell lines sensitive to and resistant to other drugs. Dihydroartemisinin (DHA) - a known artemisinin derivative, its anti-cancer activity has been widely studied by scholars throughout the world over the years. The antitumor effects of the tumor cells, such as glioma cells, breast cancer cells, colon cancer cells, lung cancer cells, ovarian cancer cells, and pancreatic cancer cells, have also been shown. A large number of studies have shown that the deletion or UNEXPRESSION of p53 genes in many tumor cells is related to the drug resistance of chemotherapy. Combined cisplatin (Cis) related research on the H1299 cell line of p53 deficient human lung adenocarcinoma. This experiment was designed to study the synergism and potential mechanism of H1299 cell apoptosis induced by dihydroartemisinin combined with cisplatin. Methods: 1 the human lung adenocarcinoma H1299 cells were cultured in vitro and the cells were divided into four groups: dihydroartemisia Artemisia. DHA group, cisplatin (Cis) group, combination group and control group, using four methyl azazazolium salt / thiazolium (MTT) test to evaluate the change of H1299 cell inhibition rate between different treatment groups.2 using p53 gene normal expression of A549 cells as control, MTT experiment compared under the same drug treatment conditions, DHA and Cis respectively to A549 fine Effect of cell and H1299 cell inhibition rate on.3 using flow cytometry (FCM) to evaluate the different time of DHA action. In the case of dose, the effect of.4 on the apoptosis rate of H1299 cells by Hearst 33258 (Hoechst 33258) to observe the morphological changes of H1299 nuclei after DHA treated.5 through Western blot (Western Blot) and inverse Transcriptional real-time quantitative polymerase chain reaction (RT-q PCR) to evaluate the expression of apoptosis related pathway proteins and genes. Results: 1 MTT and Hoechst 33258 cell nuclear staining experiments showed that the apoptosis rate of dihydroartemisinin and cisplatin was significantly increased with the increase of the concentration of each drug and the prolongation of the action time. The rate of apoptosis in the treatment group was higher. Another MTT experiment showed that the inhibition rate of A549 cells was significantly higher than that of H1299 cells after 48 hours of incubation of A549 and H1299 two groups with the same concentration of Cis. On the contrary, the inhibition rates of the two groups were both in the two groups after 48 hours of incubating the two groups of cells with the same concentration of DHA. The results of 2 flow cytometry showed that when the concentration of DHA was 20 mu M, the apoptosis rate of H1299 cells increased from 9.09% (control group) to 17.07% (24 hours) and 24.31% (48 hours); when DHA acted at the same time (24 hours), the apoptosis rate of cells increased from 9.09% (control group) to 17.07% (concentration of 20 mu M) and 22.5% (concentration 40). The results of 3 RT-q PCR showed that the single drug DHA (20 mu M), Cis (12.5 M) and two drugs were incubated for 24 hours and 48 hours. The expression of cystine aspartic proteinase (Caspase) -3, -8, -9m (35) (35) in H1299 cells was up to be higher than that of the single drug group, while the single drug group (35) (35) was higher than the single drug group. In the control group, the results of 4 Western Blot showed that the combination group could significantly improve the activity of Caspase-8, -9, -3, -6, -7 and poly (adenosine two phosphate ribose polymerase). After adding Caspase-3, -8, -9 inhibitors to H1299 cells, the expression of these three proteins was observed again. The expression of Cleavecaspase-3 was obviously reduced in K group, but the expression of Cleave-caspase-8, -9 was not obviously changed, but the expression of Cleave-caspase-8 in early Z IETD-fmk group was significantly reduced, and the expression of Cleave-caspase-3 and -9 was partly reduced. The expression of cell Cleave-caspase-9 in Z LEHD-fmk group was significantly reduced. The expression of ave-caspase-3 was reduced and the expression of Cleave-caspase-8 was basically unchanged. Conclusion: 1 DHA can induce time and dose dependent cytotoxicity of H1299 cells to.2 DHA, which can induce time and dose dependent apoptosis of H1299 cells,.3 DHA joint Cis is responsible for synergistic apoptosis of H1299 cells. -8 participates in the death receptor signaling pathway and the synergistic effect of the mitochondrial signaling pathway involved in Caspase-9 and the combination of DHA and CIS associated with.5 DHA and Cis monotherapy and the combination of Cis can up regulate the expression of Caspase-3, -8, -9m RNA. Synergistic effect with death receptor apoptosis pathway and Caspase-9 involved in mitochondrial apoptotic pathway induces apoptosis in H1299 cells.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2

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