Wnt信號通路激活Caski細胞系Twist基因的檢測與分析
發(fā)布時間：2018-10-08 13:37
【摘要】：目的：通過氯化鋰激活宮頸癌Caski細胞Wnt信號通路后，檢測Twist基因的mRNA和蛋白水平表達差異，探討宮頸癌Caski細胞系Wnt信號通路與Twist基因的關(guān)系，為宮頸癌的發(fā)病機制提供實驗依據(jù)。 方法：體外培養(yǎng)宮頸癌Caski細胞，不同濃度的氯化鋰激活Wnt信號通路后，MTT篩選出適宜的激活劑濃度，實驗分為7組：對照組（未加氯化鋰的Caski細胞）及氯化鋰濃度分別為0.8、0.4、0.2、0.1、0.05、0.025mol/L，，分別培養(yǎng)12h、24h， MTT檢測各組細胞在不同濃度和時間點細胞生長抑制率，篩選出氯化鋰的最低抑制濃度和第二低抑制濃度；Western blot檢測Wnt信號通路活化的標志蛋白β-catenin的表達水平。RT-PCR和Western blot檢測Caski細胞系在Wnt信號通路激活狀態(tài)Twist基因的mRNA和蛋白表達水平；實驗分為5組：（1）對照組（未加氯化鋰的Caski細胞）；（2）氯化鋰最低抑制濃度處理12h；（3）氯化鋰最低抑制濃度處理24h；（4）氯化鋰第二低抑制濃度處理12h；（5）氯化鋰第二低抑制濃度處理24h。RT-PCR和Western-blot檢測五組細胞中Twist基因的表達情況，分析Wnt信號通路激活狀態(tài)下Twist基因的mRNA和蛋白的表達水平。 結(jié)果：1.不同濃度氯化鋰處理Caski細胞，在培養(yǎng)12h和24h后，計算得出氯化鋰最低抑制濃度和第二低抑制濃度分別為0.05mol/L、0.1mol/L，兩組濃度在12h、24h相對應(yīng)的細胞抑制率為6.600±0.027，4.200±0.063和21.800±0.021，46.200±0.073。 2.氯化鋰激活Wnt信號通路后，β-catenin的表達水平增高（P0.01）。 3.氯化鋰0.05mol/L、0.1mol/L分別處理12h組，氯化鋰0.05mol/L、0.1mol/L分別處理24h組中Twist基因的mRNA和蛋白表達水平較對照組均顯著升高(P0.05)，表明Caski細胞系中Wnt信號通路激活后，可以導致Twist基因的表達增高。 結(jié)論： 1.氯化鋰作用于Caski細胞后，β-catenin的表達水平增高，說明Wnt信號通路激活。 2.在Caski細胞中，氯化鋰通過激活Wnt信號通路而促進Twist基因的表達上調(diào)。
[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 鹵0.027 鹵4.200 鹵0.063 and 21.800 鹵0.021 鹵0.021 鹵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.
【學位授予單位】：南華大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R737.33
本文編號：2256947
[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 鹵0.027 鹵4.200 鹵0.063 and 21.800 鹵0.021 鹵0.021 鹵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.
【學位授予單位】：南華大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R737.33
【參考文獻】
相關(guān)期刊論文 前3條
1 張建;顧茵;羅學勝;楊忠;李紅麗;蔡文琴;;氯化鋰通過激活Wnt信號通路促進神經(jīng)干細胞增殖[J];濟寧醫(yī)學院學報;2013年06期
2 汪雯雯;栗妍;陶濤;王恬;楊潤峰;吳章穎;王世宣;;TWIST介導的上皮-間質(zhì)轉(zhuǎn)化與宮頸癌發(fā)生發(fā)展相關(guān)性的研究[J];中華腫瘤防治雜志;2011年22期
3 王恬;陶濤;阿比丹·吐爾汗江;汪雯雯;栗妍;王世宣;;Twist介導的衰老逃逸與宮頸癌侵襲轉(zhuǎn)移的關(guān)系[J];華中科技大學學報(醫(yī)學版);2013年06期
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