Wnt信号通路激活Caski细胞系Twist基因的检测与分析

发布时间：2018-10-08 13:37

【摘要】：目的：通过氯化锂激活宫颈癌Caski细胞Wnt信号通路后，检测Twist基因的mRNA和蛋白水平表达差异，探讨宫颈癌Caski细胞系Wnt信号通路与Twist基因的关系，为宫颈癌的发病机制提供实验依据。方法：体外培养宫颈癌Caski细胞，不同浓度的氯化锂激活Wnt信号通路后，MTT筛选出适宜的激活剂浓度，实验分为7组：对照组（未加氯化锂的Caski细胞）及氯化锂浓度分别为0.8、0.4、0.2、0.1、0.05、0.025mol/L，，分别培养12h、24h， MTT检测各组细胞在不同浓度和时间点细胞生长抑制率，筛选出氯化锂的最低抑制浓度和第二低抑制浓度；Western blot检测Wnt信号通路活化的标志蛋白β-catenin的表达水平。RT-PCR和Western blot检测Caski细胞系在Wnt信号通路激活状态Twist基因的mRNA和蛋白表达水平；实验分为5组：（1）对照组（未加氯化锂的Caski细胞）；（2）氯化锂最低抑制浓度处理12h；（3）氯化锂最低抑制浓度处理24h；（4）氯化锂第二低抑制浓度处理12h；（5）氯化锂第二低抑制浓度处理24h。RT-PCR和Western-blot检测五组细胞中Twist基因的表达情况，分析Wnt信号通路激活状态下Twist基因的mRNA和蛋白的表达水平。结果：1.不同浓度氯化锂处理Caski细胞，在培养12h和24h后，计算得出氯化锂最低抑制浓度和第二低抑制浓度分别为0.05mol/L、0.1mol/L，两组浓度在12h、24h相对应的细胞抑制率为6.600±0.027，4.200±0.063和21.800±0.021，46.200±0.073。 2.氯化锂激活Wnt信号通路后，β-catenin的表达水平增高（P0.01）。 3.氯化锂0.05mol/L、0.1mol/L分别处理12h组，氯化锂0.05mol/L、0.1mol/L分别处理24h组中Twist基因的mRNA和蛋白表达水平较对照组均显著升高(P0.05)，表明Caski细胞系中Wnt信号通路激活后，可以导致Twist基因的表达增高。结论： 1.氯化锂作用于Caski细胞后，β-catenin的表达水平增高，说明Wnt信号通路激活。 2.在Caski细胞中，氯化锂通过激活Wnt信号通路而促进Twist基因的表达上调。
[Abstract]:Objective: to investigate the relationship between the expression of mRNA and protein of Twist gene and the expression of Wnt signal pathway in cervical cancer Caski cell line, and to explore the relationship between Wnt signaling pathway and Twist gene after activation of Wnt signal pathway by lithium chloride in cervical cancer Caski cells. To provide experimental evidence for the pathogenesis of cervical cancer. Methods: cervical cancer Caski cells were cultured in vitro. After different concentrations of lithium chloride activated Wnt signaling pathway, the suitable concentration of activator was selected. The experiment was divided into 7 groups: the control group (Caski cells without lithium chloride) and the concentration of lithium chloride were 0.8g / L 0.4g / L 0.1g / L 0.05U 0.025mol / L, cultured for 12h / 24h, respectively. MTT was used to detect the cell growth inhibition rate at different concentrations and time points. The expression level of 尾 -catenin, a marker of activation of Wnt signaling pathway, was detected by Western blot and Western blot was used to detect the mRNA and protein expression of Twist gene in Wnt signaling pathway. The experiment was divided into five groups: (1) the control group (); (_ 2 without lithium chloride) was treated with lithium chloride for 12 h; (3) the lowest inhibitory concentration of lithium chloride was treated for 24 h; (4) the second low inhibitory concentration of lithium chloride was treated for 12 h; (5) 24h.RT-PCR and Western-blot were used to detect the expression of Twist gene in five groups of cells, and the expression level of mRNA and protein of Twist gene in the activated state of Wnt signaling pathway was analyzed. The result is 1: 1. Caski cells were treated with different concentrations of lithium chloride for 12 h and 24 h. The results showed that the lowest and second lowest inhibitory concentrations of lithium chloride were 0.05mol / L 0.1 mol / L and 6.600 卤0.027 卤4.200 卤0.063 and 21.800 卤0.021 卤0.021 卤0.073, respectively. 2. The expression of 尾-catenin increased after lithium chloride activated the Wnt signaling pathway (P0.01). 3. The mRNA and protein expression of Twist gene in the treatment group of lithium chloride 0.05mol / L 0.1 mol / L for 12 h and the group treated with lithium chloride 0.05mol / L 0.1 mol / L for 24 h were significantly higher than those in the control group (P0.05), which indicated that the expression of Twist gene was increased after the activation of Wnt signaling pathway in Caski cell line. Conclusion: 1. The expression of 尾 -catenin in Caski cells was increased after treated with lithium chloride, indicating that Wnt signaling pathway was activated. 2. In Caski cells, lithium chloride promotes the up-regulation of Twist gene expression by activating the Wnt signaling pathway.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.33

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