强心苷类药物对结肠癌细胞的抑制作用及体外机制研究

发布时间：2018-04-24 00:09

本文选题：强心苷 + Oleandrin　；参考：《南方医科大学》2017年硕士论文

【摘要】：研究背景强心苷类药物包括oleandrin,对许多不同类型的癌细胞具有细胞毒性作用,在我们前期的高通量筛选中,发现一些强心苷类药物能够特异性地杀死人结肠癌细胞。其中,oleandrin就是一种植物来源于夹竹桃的强心苷类药物,其抗肿瘤作用机制仍有很多方面并没有被清楚的阐释。本文便以oleandrin对人结肠癌细胞的作用及潜在的机制进行探讨。目的本文主要通过体外研究oleandrin对人结肠癌细胞生长的影响及对SW480细胞的潜在机制,为体外研究强心苷药物治疗人结肠癌提供理论基础。方法1.MTT法对强心苷类小分子化合物IC50的测定。2.CCK-8法检测Oleandrin对结肠癌细胞的体外抑制作用:检测对SW480细胞以及人正常上皮细胞NCM460的细胞活性。测定在孵育不同时间下对结肠癌SW480细胞生长抑制作用;对HCT116,RKO,SW480不同结肠癌细胞的影响;以及5-氟尿嘧啶IC50值的测定。3.Oleandrin对结肠癌细胞的体外诱导凋亡作用机制的研究:以Annexin V-FITC和PI标记的试剂,检测细胞周期、凋亡情况;钙离子荧光探针Fluo-3,AM检测结肠癌细胞内Ca2+浓度的变化;Caspase-3,9活性检测试剂盒来考察Caspase-3,9的活化程度;蛋白免疫印迹分析法(Western blot)对相关凋亡蛋白cytC,BAX,BCL-2,Caspase-3,9的表达情况检测;谷胱甘肽(GSH)检测试剂盒检测细胞内GSH含量。结果1.在5个强心苷类小分子化合物中,oleandrin的IC50值最低(0.02μM),处理24h后,高剂量的oleandrin并没有显著影响结肠上皮细胞的活性(IC50=0.56μM)。2.Oleandrin显著抑制SW480细胞生长,具有浓度依赖关系;处理时间24h与48 h、72 h两组相比,有显著性差异(p0.05),而48、72 h处理时间组相比,无显著性差异,各组与阴性对照组相比,均存在显著性差异(p0.01,p0.001)。Oleandrin对RKO/SW480/HCT116细胞的生长抑制作用呈剂量依赖性(p0.01,p0.001)。其 IC50 值显著低于 5-FU(IC50=22.75μM)。3.Oleandrin对结肠癌细胞能够阻滞G2/M期,并诱导凋亡,与阴性对照组相比,具有显著性差异(p0.05,p0.01);能够增加胞内Ca2+水平:在0.02μM的oleandrin处理SW480细胞后,能够时间依赖性增加细胞内活化的Caspase-3,9水平;Western blot显示能够上调cytC,BAX蛋白表达,下调BCL-2,Procaspase-3,9蛋白的表达;与阴性对照组相比,具有显著性差异(p0.05,p0.001);能剂量依赖性的降低SW480细胞内的GSH水平。结论1.Oleandrin在5种小分子化合物中IC50值最低,为0.02μM。在48h,作用抑制率达最大值;对HCT116,RKO在内的其它结肠癌细胞同样抑制生长;其IC50值明显低于5-FU。2.Oleandrin能阻滞SW480和RKO细胞G2/M期,诱导早期凋亡;具有时间及浓度依赖性的增加细胞内Ca2+水平;呈时间依赖性增加细胞内活化的Caspase-3,9水平,且在8h后,活化的Caspase-3水平达到最大值;蛋白表达检测说明其引起凋亡的过程与细胞线粒体信号通道和凋亡酶的激活途径关联。能够浓度依赖地降低SW480细胞内的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.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96

【参考文献】