盐霉素对肾上腺皮质癌SW13细胞增殖、凋亡的影响及其机制探讨

发布时间：2018-03-14 18:08

本文选题：盐霉素　切入点：肾上腺皮质癌　出处：《南方医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:探讨盐霉素在体内外对肾上腺皮质癌SW13细胞的增殖、凋亡、周期、侵袭的影响及其作用机制。方法:以浓度为30、20、10、5、2.5、1.25、0.625、0μmol/L的盐霉素处理SW13细胞,分别在24、48、72、96h时用Cell Counting Kit-8(CCK-8)试剂盒检测其对增殖的影响并计算半抑制浓度(IC50)。流式细胞仪检测盐霉素处理后SW13细胞的周期及凋亡。通过AO-EB染色在荧光显微镜下观察盐霉素处理后SW13细胞的凋亡情况。划痕实验检测盐霉素对SW13细胞迁移能力的影响。Transwell侵袭实验检测盐霉素对SW13细胞侵袭力的影响。蛋白免疫印记法检测β-链蛋白(β-catenin)与B细胞淋巴瘤/白血病-2蛋白(Bcl-2)的表达。体内实验观察盐霉素对裸鼠移植瘤生长的抑制情况。TUNNEL实验检测盐霉素在体内对SW13细胞凋亡的影响。免疫组化实验观察盐霉素在体内对SW13移植瘤PCNA、Bcl-2、β-catenin蛋白表达水平的影响。结果:CCK-8结果显示0～30μmol/L的盐霉素对SW13细胞的抑制呈明显的时间-计量依赖效应,24、48、72、96h对应的IC50依次为25.65、7.97、2.89、0.40μmol/L。流式检测结果显示,盐霉素处理SW13细胞后处于G1期的细胞百分比分别为(66.250±1.162)%、(71.693±0.849)%,与对照组(59.837±3.043)%相比差异具有统计学意义(P0.05);凋亡率分别为9.9%、30.3%、3.4%,差异具有统计学意义(P0.05)。AO-EB染色在荧光显微镜下观察盐霉素处理后SW13细胞的凋亡率较对照组明显升高。细胞划痕实验结果显示盐霉素SW13细胞的迁移能力下降。Transwell结果显示对照组与实验组细胞侵袭数分别为66.800±5.263、30.400±2.881、17.600±3.050,差异有统计学意义(P0.05)。蛋白质免疫印迹结果显示Wnt/β-catenin信号通路相关蛋白β-catenin及抑制凋亡相关蛋白Bcl-2表达下调。体内实验结果表明盐霉素可抑制裸鼠移植瘤的生长。TUNNEL实验表明盐霉素在体内也可促进SW13细胞的凋亡。移植瘤免疫组化实验结果表明盐霉素在体内可降低SW13移植瘤PCNA、Bcl-2、β-catenin蛋白表达水平。结论:盐霉素在体内外可抑制肾上腺皮质癌SW13细胞的增殖,阻滞其周期,减弱侵袭力并诱导凋亡,其机制与干扰Wnt/β-catenin信号通路及下调PCNA及凋亡抑制蛋白Bcl-2有关。
[Abstract]:Objective: to investigate the effects of salinomycin on the proliferation, apoptosis, cycle and invasion of adrenal cortical carcinoma (SW13) cells in vitro and in vivo. The effect of Cell Counting Kit-8 CCK-8 on proliferation was detected by Cell Counting Kit-8 CCK-8 kit and the semi-inhibitory concentration was calculated at 96 h. Flow cytometry was used to detect the cell cycle and apoptosis of SW13 cells treated with salinomycin. The AO-EB staining was used to observe the effect of salinomycin on the proliferation of SW13 cells. The effect of salinomycin on the migration of SW13 cells was detected by scratch assay. The effect of salinomycin on the invasiveness of SW13 cells was detected by transwell invasion assay. The 尾 -catenin and B thin cells were detected by protein imprinting assay. The effect of salinomycin on the growth of xenografts in nude mice was observed in vivo. The effect of salinomycin on the apoptosis of SW13 cells in vivo was detected by Tunel assay. The effect of salinomycin on the apoptosis of SW13 cells was observed by immunohistochemistry. Results the inhibitory effect of 0 ~ 30 渭 mol/L salinomycin on SW13 cells was time-metrological dependent in vivo, and the corresponding IC50 for 96 h was 25.657.97.72.89 渭 mol / L and 0.40 渭 mol 路L ~ (-1), respectively. The percentage of cells in G1 phase of SW13 treated with salinomycin was 66.250 卤1.162 and 71.693 卤0.849, respectively, which was significantly higher than that of control group (59.837 卤3.043%), and the apoptotic rate was 9.90.33.4.The difference was statistically significant under fluorescence microscope. The cell scratch test showed that the migration ability of salinomycin SW13 cells decreased. Transwell results showed that the number of cell invasion in the control group and the experimental group was 66.800 卤5.263 and 30.400 卤2.881or 17.600 卤3.050, respectively. The difference was statistically significant. Wnt/ 尾 -catenin signaling pathway related protein 尾 -catenin and inhibiting apoptosis-related protein Bcl-2 expression were down-regulated. In vivo, salinomycin could inhibit the growth of xenografts in nude mice. The results of immunohistochemistry showed that salinomycin could decrease the expression of PCNA Bcl-2, 尾 -catenin protein in SW13 xenografts. Conclusion: salinomycin can inhibit the proliferation of SW13 cells in adrenal cortical carcinoma in vivo and in vitro. The mechanism is related to the interference of Wnt/ 尾 -catenin signaling pathway and down-regulation of PCNA and Bcl-2.
【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】：R736.6

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