本文選題：雙氫青蒿素 + 順鉑�。� 參考：《河北醫(yī)科大學》2017年碩士論文

【摘要】：目的:肺癌的發(fā)病率及相關(guān)死亡率在目前全球癌癥中所占比例最高。其中80%的肺癌病人為非小細胞肺癌(NSCLC)。目前臨床一線使用的含鉑化療方案有效率僅有30%。因此,大家迫切的需要找到一種新的化療藥物來提高肺癌病人的治療效率。近年的許多研究中,青蒿素及其衍生物在一些對其他藥物敏感及耐藥的腫瘤細胞株中均展現(xiàn)出了強大的抗癌活性。雙氫青蒿素(DHA)—一種已知的青蒿素的衍生物,這些年來,它的抗癌活性在世界范圍內(nèi)被各國學者廣泛研究。而雙氫青蒿素也展現(xiàn)出了對腫瘤細胞株如神經(jīng)膠質(zhì)瘤細胞、乳腺癌細胞、結(jié)腸癌細胞、肺癌細胞、卵巢癌細胞、胰腺癌細胞的抗癌作用。大量的研究表明許多腫瘤細胞p53基因的缺失或未表達均與化療的耐藥有關(guān)。但迄今為止,尚未查到關(guān)于雙氫青蒿素聯(lián)合順鉑(Cis)作用于p53缺失型人肺腺癌H1299細胞系的相關(guān)研究。而這項實驗的設(shè)計初衷就是研究雙氫青蒿素聯(lián)合順鉑誘導H1299細胞凋亡的協(xié)同作用及其潛在的作用機制。方法:1體外培養(yǎng)人類肺腺癌H1299細胞并將細胞分為四組:雙氫青蒿素(DHA)組、順鉑(Cis)組、聯(lián)合用藥組及對照組,通過四甲基偶氮唑鹽/噻唑藍(MTT)實驗來評價不同處理組間H1299細胞抑制率的變化情況。2采用p53基因正常表達的A549細胞作為對照,通過MTT實驗比較在相同藥物處理條件下,DHA及Cis分別對A549細胞和H1299細胞抑制率的影響。3采用流式細胞術(shù)(FCM)來評價DHA作用不同時間、劑量的情況下,對H1299細胞的凋亡率的影響。4通過赫斯特33258(Hoechst 33258)對細胞核染色來觀察經(jīng)DHA處理后H1299細胞核的形態(tài)變化。5通過蛋白印跡法(Western Blot)和逆轉(zhuǎn)錄實時定量聚合酶鏈反應(yīng)(RT-q PCR)來評價凋亡相關(guān)通路蛋白及基因的表達情況。結(jié)果:1 MTT實驗和Hoechst 33258細胞核染色實驗顯示隨著各自藥物濃度的增加以及作用時間的延長雙氫青蒿素和順鉑單藥處理組細胞凋亡率明顯增加,聯(lián)合用藥處理組的細胞凋亡率更高。另一個MTT實驗結(jié)果顯示:相同濃度的Cis孵育A549及H1299兩組細胞48小時后,對A549細胞的抑制率明顯高于對H1299細胞的抑制率。于此相反,使用相同濃度的DHA孵育上述兩組細胞48小時后,兩組間的細胞抑制率均無明顯差異;2流式細胞術(shù)的結(jié)果顯示當DHA濃度為20μM時,H1299細胞的凋亡率從9.09%(對照組)上升至17.07%(24小時)以及24.31%(48小時);當DHA作用于相同時間(24小時),其引起細胞的凋亡率從9.09%(對照組)上升至17.07%(濃度為20μM)以及22.5%(濃度為40μM);3 RT-q PCR的結(jié)果顯示單藥DHA(20μM)、Cis(12.5μM)及兩藥聯(lián)合孵育24小時及48小時,H1299細胞中半胱天冬氨酸蛋白酶(Caspase)-3、-8、-9m RNA的表達均上調(diào),其中聯(lián)合用藥組Caspase-3,-8,-9的2-(35)(35)Ct值高于單藥組,而單藥組Caspase3,8,9的2-(35)(35)Ct值高于對照組;4 Western Blot結(jié)果顯示聯(lián)合用藥組能顯著提高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細胞中加入Caspase-3、-8、-9抑制劑后,再次通過Western Blot實驗觀察這三個蛋白的表達情況,結(jié)果表明提前加入z DQMD-fmk組的細胞,Cleavecaspase-3的表達明顯降低,但是Cleave-caspase-8、-9的表達無明顯變化。而提前加入z IETD-fmk組的細胞,顯著降低了Cleave-caspase-8的表達,部分降低了Cleave-caspase-3、-9的表達。提前孵育z LEHD-fmk組的細胞Cleave-caspase-9的表達顯著降低,Cleave-caspase-3的表達部分降低,而Cleave-caspase-8的表達基本無變化。結(jié)論:1 DHA作用于H1299細胞能誘導時間及劑量依賴性細胞毒性。2 DHA作用于H1299細胞能誘導時間及劑量依賴性的細胞凋亡。3 DHA聯(lián)合Cis作用于H1299細胞有協(xié)同促凋亡作用。4 Caspase-8參與的死亡受體信號通路及Caspase-9參與的線粒體信號通路與DHA及CIS聯(lián)合用藥產(chǎn)生的協(xié)同作用有關(guān)。5 DHA及Cis單藥及聯(lián)合用藥均能上調(diào)H1299細胞Caspase-3,-8,-9m RNA的表達。綜上所述,本研究結(jié)果表明雙氫青蒿素聯(lián)合順鉑可能通過Caspase-8參與的死亡受體凋亡通路和Caspase-9參與的線粒體凋亡通路產(chǎn)生協(xié)同作用誘導H1299細胞凋亡。
[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.
【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R734.2

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