本文選題：強心苷 + Oleandrin　； 參考：《南方醫(yī)科大學(xué)》2017年碩士論文

【摘要】：研究背景強心苷類藥物包括oleandrin,對許多不同類型的癌細(xì)胞具有細(xì)胞毒性作用,在我們前期的高通量篩選中,發(fā)現(xiàn)一些強心苷類藥物能夠特異性地殺死人結(jié)腸癌細(xì)胞。其中,oleandrin就是一種植物來源于夾竹桃的強心苷類藥物,其抗腫瘤作用機制仍有很多方面并沒有被清楚的闡釋。本文便以oleandrin對人結(jié)腸癌細(xì)胞的作用及潛在的機制進(jìn)行探討。目的本文主要通過體外研究oleandrin對人結(jié)腸癌細(xì)胞生長的影響及對SW480細(xì)胞的潛在機制,為體外研究強心苷藥物治療人結(jié)腸癌提供理論基礎(chǔ)。方法1.MTT法對強心苷類小分子化合物IC50的測定。2.CCK-8法檢測Oleandrin對結(jié)腸癌細(xì)胞的體外抑制作用:檢測對SW480細(xì)胞以及人正常上皮細(xì)胞NCM460的細(xì)胞活性。測定在孵育不同時間下對結(jié)腸癌SW480細(xì)胞生長抑制作用;對HCT116,RKO,SW480不同結(jié)腸癌細(xì)胞的影響;以及5-氟尿嘧啶IC50值的測定。3.Oleandrin對結(jié)腸癌細(xì)胞的體外誘導(dǎo)凋亡作用機制的研究:以Annexin V-FITC和PI標(biāo)記的試劑,檢測細(xì)胞周期、凋亡情況;鈣離子熒光探針Fluo-3,AM檢測結(jié)腸癌細(xì)胞內(nèi)Ca2+濃度的變化;Caspase-3,9活性檢測試劑盒來考察Caspase-3,9的活化程度;蛋白免疫印跡分析法(Western blot)對相關(guān)凋亡蛋白cytC,BAX,BCL-2,Caspase-3,9的表達(dá)情況檢測;谷胱甘肽(GSH)檢測試劑盒檢測細(xì)胞內(nèi)GSH含量。結(jié)果1.在5個強心苷類小分子化合物中,oleandrin的IC50值最低(0.02μM),處理24h后,高劑量的oleandrin并沒有顯著影響結(jié)腸上皮細(xì)胞的活性(IC50=0.56μM)。2.Oleandrin顯著抑制SW480細(xì)胞生長,具有濃度依賴關(guān)系;處理時間24h與48 h、72 h兩組相比,有顯著性差異(p0.05),而48、72 h處理時間組相比,無顯著性差異,各組與陰性對照組相比,均存在顯著性差異(p0.01,p0.001)。Oleandrin對RKO/SW480/HCT116細(xì)胞的生長抑制作用呈劑量依賴性(p0.01,p0.001)。其 IC50 值顯著低于 5-FU(IC50=22.75μM)。3.Oleandrin對結(jié)腸癌細(xì)胞能夠阻滯G2/M期,并誘導(dǎo)凋亡,與陰性對照組相比,具有顯著性差異(p0.05,p0.01);能夠增加胞內(nèi)Ca2+水平:在0.02μM的oleandrin處理SW480細(xì)胞后,能夠時間依賴性增加細(xì)胞內(nèi)活化的Caspase-3,9水平;Western blot顯示能夠上調(diào)cytC,BAX蛋白表達(dá),下調(diào)BCL-2,Procaspase-3,9蛋白的表達(dá);與陰性對照組相比,具有顯著性差異(p0.05,p0.001);能劑量依賴性的降低SW480細(xì)胞內(nèi)的GSH水平。結(jié)論1.Oleandrin在5種小分子化合物中IC50值最低,為0.02μM。在48h,作用抑制率達(dá)最大值;對HCT116,RKO在內(nèi)的其它結(jié)腸癌細(xì)胞同樣抑制生長;其IC50值明顯低于5-FU。2.Oleandrin能阻滯SW480和RKO細(xì)胞G2/M期,誘導(dǎo)早期凋亡;具有時間及濃度依賴性的增加細(xì)胞內(nèi)Ca2+水平;呈時間依賴性增加細(xì)胞內(nèi)活化的Caspase-3,9水平,且在8h后,活化的Caspase-3水平達(dá)到最大值;蛋白表達(dá)檢測說明其引起凋亡的過程與細(xì)胞線粒體信號通道和凋亡酶的激活途徑關(guān)聯(lián)。能夠濃度依賴地降低SW480細(xì)胞內(nèi)的GSH水平。
[Abstract]:Background Cardioside drugs including oleandrinins have cytotoxic effects on many different types of cancer cells. In our previous high-throughput screening, we found that some cardioside drugs can specifically kill human colon cancer cells. Oleandrin is one of the anticardioside drugs derived from oleandrin. The mechanism of its antitumor effect has not been clearly explained in many aspects. In this paper, the effect and potential mechanism of oleandrin on human colon cancer cells were discussed. Objective to study the effect of oleandrin on the growth of human colon cancer cells and its potential mechanism on SW480 cells in vitro, and to provide a theoretical basis for the study of cardioside drugs in vitro for the treatment of human colon cancer. Methods the inhibitory effect of Oleandrin on colon cancer cells in vitro was determined by 1.MTT assay. 2. The activity of NCM460 in SW480 cells and human normal epithelial cells was detected by CCK-8 method. The inhibitory effects on the growth of colon cancer SW480 cells and the effects on HCT116 and RKOSW480 colon cancer cells were measured. The mechanism of apoptosis induced by Oleandrin on colon cancer cells in vitro was studied. The cell cycle and apoptosis were detected with Annexin V-FITC and Pi labeled reagents. The changes of Ca2 concentration in colon cancer cells were detected by calcium fluorescence probe Fluo-3AM-and the activation of Caspase-3Caspase-9 was detected by the assay kit, and the expression of apoptosis protein cyt cyt cyt BBAXCL-2 (Caspase-3O9) was detected by Western blotanalysis. Glutathione (GSH) assay kit was used to detect the content of GSH in the cells. Result 1. The IC50 value of Oleandrin was the lowest (0.02 渭 M) among the five cardiosides. After 24 hours of treatment, the high dose of oleandrin did not significantly affect the activity of colon epithelial cells. IC50 and 0.56 渭 M).2.Oleandrin significantly inhibited the growth of SW480 cells in a concentration-dependent manner. There was significant difference in the treatment time of 24 h and 48 h / 72 h, but there was no significant difference between the 48 h group and the control group. There was a significant difference between the two groups in the growth inhibition of RKO/SW480/HCT116 cells in a dose-dependent manner. The IC50 value of 5-FU(IC50=22.75 渭 M).3.Oleandrin was significantly lower than that of 5-FU(IC50=22.75 渭 M).3.Oleandrin, which could block the G _ 2 / M phase and induce apoptosis in colon cancer cells. Compared with the negative control group, the IC50 value of 5-FU(IC50=22.75 渭 M).3.Oleandrin was significantly lower than that of the negative control group, and the intracellular Ca2 level could be increased after the treatment of SW480 cells with 0.02 渭 M oleandrin. The expression of cyt Cnbax protein was up-regulated, and the expression of BCL-2 and Procaspase-3 was down-regulated by Western blot, which was significantly different from that of negative control group, and could decrease the level of GSH in SW480 cells in a dose-dependent manner. Conclusion among the five small molecular compounds, 1.Oleandrin has the lowest IC50 value (0.02 渭 m). At 48h, the inhibition rate reached the maximum, and the growth of other colon cancer cells including HCT116RKO was also inhibited. The IC50 value was significantly lower than that of 5-FU.2.Oleandrin, which could block the G _ 2 / M phase of SW480 and RKO cells and induce early apoptosis, and increase the intracellular Ca2 level in a time-and concentration-dependent manner. In a time-dependent manner, the level of activated Caspase-3 was increased, and the level of activated Caspase-3 reached its maximum after 8 hours. The expression of protein showed that the process of apoptosis was associated with mitochondrial signal channel and activation pathway of apoptotic enzyme. The level of GSH in SW480 cells was decreased in a concentration-dependent manner.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R96

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