甘草查尔酮B抑制膀胱肿瘤细胞生长及转移作用研究

发布时间：2018-05-07 08:17

本文选题：甘草查尔酮B + 膀胱癌细胞　；参考：《兰州大学》2014年博士论文

【摘要】：目的：研究甘草查尔酮B(Licochalcone B,LCB)对膀胱肿瘤细胞生长抑制及转移能力的影响,并阐明其作用机制,为甘草查尔酮B治疗膀胱肿瘤的临床应用提供理论依据。方法：四甲基偶氮唑盐实验(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide,MTT)测定甘草查尔酮B对T24、EJ细胞生长的抑制效应；Hoechst荧光染色观察细胞形态变化；流式细胞技术检测甘草查尔酮B对细胞周期和细胞凋亡率影响；实时定量PCR(Real-time quantitative PCR, qRT-PCR)检测甘草查尔酮B对细胞周期相关蛋白在mRNA水平的影响；Westernblot检测甘草查尔酮B对细胞分裂周期蛋白(CDC25A,CDC25B),以及凋亡相关蛋白Bcl-2,Bax,Caspase-3,PARP和Survivin表达的影响；采用MTT法检测甘草查尔酮B对T24细胞与基质粘附能力的影响；划痕试验测定甘草查尔酮B对T24细胞体外迁移能力的影响；采用qRT-PCR.明胶酶谱两种方法测定甘草查尔酮B对细胞基质金属蛋白酶(MMP-2,MMP-9)表达的影响；酶联免疫法(ELISA)检测甘草查尔酮B对激活蛋白-1(AP-1)、核转录因子(NF-κB)表达的影响。建立C57BL/6小鼠皮下膀胱癌模型,分离肿瘤组织,利用HE染色和Tunnel法检测甘草查尔酮B对膀胱肿瘤的体内抑瘤作用。结果：(1)经不同浓度(0,10,20,40,60,80μM)甘草查尔酮B分别处理24h,48h和72h,T24、EJ细胞增殖活力降低,当甘草查尔酮B浓度增加到20μM以上时(40,60,80μM)细胞增殖率明显低于对照组,呈浓度及时间依赖性变化；72h尤为明显,ICso分别为39.18±0.51μM,34.15±0.36μM。 (2)T24和EJ细胞经不同浓度甘草查尔酮B处理72h后,甘草查尔酮B能够诱导肿瘤细胞S期阻滞；降低S期相关周期蛋白(cyclinA)及蛋白激酶(CDK1,CDK2)的mRNA表达；降低S期相关细胞分裂周期蛋白Cdc25A.Cdc25B在蛋白水平的表达。 (3)不同浓度甘草查尔酮B处理T24和EJ细胞72h后,能够诱导细胞凋亡；下调Bcl-2表达,上调Bax和Caspase-3在蛋白水平上的表达,裂解PARP蛋白；并且在整体动物水平上能够抑制鼠膀胱肿瘤细胞增殖,诱导凋亡 (4)不同浓度甘草查尔酮B处理T24细胞24h后,细胞与基质间粘附力降低；与对照组相比,甘草查尔酮B显著降低MMP-9在mRNA及蛋白水平的表达,但对MMP-2表达无明显影响；能显著抑制NF-1cB表达,但对AP-1表达无明显影响。结论：以上研究结果说明,甘草查尔酮B具有抑制膀胱肿瘤细胞生长和转移的作用,是一种有应用前景的膀胱肿瘤灌注药物。
[Abstract]:Objective: to study the effect of B(Licochalcone BB(Licochalcone on the growth inhibition and metastasis of bladder tumor cells, and to elucidate its mechanism, and provide theoretical basis for the clinical application of Glycyrrhiza chalcone B in the treatment of bladder cancer. Methods: the inhibitory effect of Glycyrrhiza B on the growth of T24EJ cells was determined by MTT assay. The morphological changes of T24EJ cells were observed by Hoechst fluorescence staining and the effects of glycyrrhizin B on cell cycle and apoptosis were detected by flow cytometry. The effect of glycyrrhizin B on cell cycle related protein (mRNA) was detected by real-time quantitative PCR(Real-time quantitative and qRT-PCR. Western blot was used to detect the effect of glycyrrhizin B on the expression of CDC25AnCDC25B, and apoptosis related protein Bcl-2nBaxCaspase-3PARP and Survivin. The effect of glycyrrhizin B on the adhesion of T24 cells to matrix was detected by MTT assay, the effect of glycyrrhizin B on the migration ability of T24 cells in vitro was determined by scratch test, and the migration ability of T24 cells was determined by qRT-PCR. The effect of glycyrrhizin B on the expression of matrix metalloproteinase-2 (MMP-2) MMP-9 and the expression of activator protein-1 AP-1 and nuclear transcription factor-NF-魏 B were detected by enzyme linked immunosorbent assay (Elisa). The subcutaneous bladder cancer model of C57BL/6 mice was established and tumor tissues were isolated. The inhibitory effect of glycyrrhizin B on bladder cancer in vivo was detected by HE staining and Tunnel method. Results (1) the proliferation activity of T24EJ cells treated with different concentrations of 10 ~ 10 ~ (20) and 40 ~ (6080 渭 M) glycyrrhizin B for 24 h and 72 h respectively decreased. When the concentration of glycyrrhizin B increased to more than 20 渭 M, the proliferative rate of T24EJ cells was significantly lower than that of the control group, and the proliferation rate of T24EJ cells was significantly lower than that of the control group. In a concentration and time dependent manner, ICso was found to be 39.18 卤0.51 渭 m, 34.15 卤0.36 渭 M for 72 h, respectively. After treated with different concentration of glycyrrhizin B for 72 hours, T24 and EJ cells could induce S phase arrest and decrease the mRNA expression of cyclin A and CDK1 + CDK2. The expression of cyclin Cdc25A.Cdc25B in S phase related cells was decreased at the protein level. T24 and EJ cells treated with different concentration of glycyrrhizin B for 72 h could induce apoptosis, down-regulate the expression of Bcl-2, up-regulate the expression of Bax and Caspase-3 at protein level, and break down the PARP protein. And it can inhibit the proliferation of bladder tumor cells and induce apoptosis on the whole animal level. (4) the adhesion of T24 cells treated with different concentration of glycyrrhizin B decreased after 24 hours, compared with the control group, glycyrrhizin B significantly decreased the expression of MMP-9 in mRNA and protein levels, but had no significant effect on the expression of MMP-2. It could significantly inhibit the expression of NF-1cB, but had no effect on the expression of AP-1. Conclusion: these results suggest that glycyrrhiza chalcone B can inhibit the growth and metastasis of bladder tumor cells and is a promising drug for bladder tumor perfusion.
【学位授予单位】：兰州大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.14

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