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Anti-apoptosis Effect of Decoy Receptor 3 in Cholangiocarcinoma Cell Line tfk-1

Chinese Medical Journal 2018年第1期131卷 82-87页

作者： Ying-Chen Xu Jing Cui Li-Jun Zhang Dong-Xin Zhang Bing-Chen Xing Xiong-Wei-Ye Huang Ji-Xiang Wu Chao-Jie Liang Guang-Ming Li Department of Hepatobiliary Surgery Surgical Laboratory Beijing Tong Ren Hospital Capital Medical University Beijing 100730 China Department of Pathology Capital Medical University Beijing 100069 China

Background： Decoy receptor 3 （DcR3） is a protein with anti-apoptotic effect that belongs to the tumor necrosis factor receptor superfamily. DcR3 is highly expressed in a variety of malignant tumors including cholangiocarcinoma and its expression was found to be related to the clinical stage, the invasion, and tile metastasis of the tumor. This in vitro study aimed to investigate the effect ofdownregulated expression of DcR3 on cell viability, cell apoptosis, and cell cycle in cholangiocarcinoma cell line tfk-1. Methods： Three different cell lines were cultured： human cholangiocarcinoma tfk-I, human biliary epithelial carcinoma HuCCT-1, and human cholangiocarcinoma RBE. The cholangiocarcinoma cell line with the highest expression of DcR3 was selected for further investigation. The expression of DcR3 was silenced/knocked down by transfection with DcR3-siRNA in the selected cell line. Various biological phenotype parameters such as cell viability, apoptosis, and cell cycle were observed. Results： The mRNA and protein levels of DcR3 were measured in the three cell lines, and tfk-1 was selected. After the treatment with DcR3-siRNA for 48 h, DcR3 mRNA and protein expression in the treatment group were 38.45% （P 〈 0.01 ） and 48.03% （P 〈 0.05） of that of＇the control, respectively. It was found that the cell viability decreased to 61.87% of the control group （P 〈 0.01 ） after the downregulation of DcR3 in cholangiocarcinoma cell line tfk- 1 by transfection with DcR3-siRNA, while tile percentage ofapoptotic cells was 2.98 times as compared with the control group （P 〈 0.05）. Compared with the control group the ratio of G0/G1 increased, and the ratio of G2/M dccreased in the treatment group. However, the differences were not statistically significant. Conclusions： The effect of DcR3 on the growth and apoptosis ofcholangiocarcinoma has been demonstrated. DcR3 is not only a predictive marker for malignant tumor but it is also likely to be a potential target for cancer gene therap

关键词： Apoptosis: Cell Cycle: Cholangiocarcinoma Decoy Receptor 3 tfk-1

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9-硝基喜树碱脂质体对胆管癌tfk-1细胞周期和凋亡的影响

9-硝基喜树碱脂质体对胆管癌TFK-1细胞周期和凋亡的影响

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作者：卢进利华中科技大学

学位级别：硕士

目的研究9-硝基喜树碱脂质体(9-nitrocamptothecin loaded liposomes,9-NC-LP)及9-硝基喜树碱( 9-nitrocamptothecin ,9-NC)对胆管癌细胞tfk-1的细胞周期和凋亡的影响及作用机制,同时对脂质体作为9-NC纳米载体的可行性进行评价。方法用... 详细信息

目的研究9-硝基喜树碱脂质体(9-nitrocamptothecin loaded liposomes,9-NC-LP)及9-硝基喜树碱( 9-nitrocamptothecin ,9-NC)对胆管癌细胞tfk-1的细胞周期和凋亡的影响及作用机制,同时对脂质体作为9-NC纳米载体的可行性进行评价。方法用不同浓度的9-NC和9-NC-LP(0ng/ml、0.4ng/ml、4ng/ml、40ng/ml、400ng/ml、4000ng/ml)细胞培养液孵育胆管癌细胞tfk-1 24h、48h、72h,然后MTT法测细胞抑制率,用不同浓度的9-NC和9-NC-LP(0ng/ml 40ng/ml、80 ng/ml、160ng/ml、320ng/ml、640ng/ml)细胞培养液孵育tfk-1胆管癌细胞24h、48h、72h,利用流式细胞仪检测的周期的分布及凋亡率,利用western blotting检测CDK2、Cyclin D、Cyclin E、Cyclin A、P27、P53、Caspase-3、Caspase-8、Caspase-9、线粒体相关凋亡诱导因AIFM-1、Bax及Bcl-2的表达变化。结果孵育24h、48h后,9-NC组和9-NC-LP组对tfk-1的抑制率没有显著性差异,但孵育于72h后,9-NC-LP组对tfk-1的抑制率明显高于9-NC组。用9-NC和9-NC-LP孵育tfk-1 24小时后,9-NC对tfk-1引起S期的阻滞作用明显强于9-NC-LP,但孵育24小时和72小时后,9-NC对tfk-1引起S期的阻滞作用明显弱于9-NC-LP。当孵育72h后,9-NC-LP组Annexin V+PI-细胞数明显高于9-NC组。用9-NC和9-NC-LP孵育tfk-1 24小时后,cyclin A( F=20.20 ,P= 0.02)、P27(F=5.12 ,P=0.05 )、P53(F=101.85 ,P1)、caspase-3( F=25.37 ,P1)、caspase-9( F=11.42 ,P= 0.01)、Bax/Bcl-2( F=39.94 ,P1)的表达三组间有显著性差异。用9-NC和9-NC-LP孵育tfk-1 72小时后,cyclin A( F=310.66,P1)、cyclin E( F=8.26,P= 0.02)、cyclin D(F=396.03,P1)、P27( F=7.50,P= 0.02)、P53( F=944.62,P1)caspase-3( F=6.93,P=0.03)、caspase-8( F=7.01 ,P=0.03)、AIFM-1( F=7.54,P=0.02 )、Bax/Bcl-2 ( F=11.95 ,P=0.01)的表达三组间有显著性差异。结论9-NC-LP对tfk-1的生长和增殖有较强抑制作用。当孵育24小时后,9-NC-LP对胆管癌细胞tfk-1细胞周期S期阻滞和周期相关蛋白的影响弱于9-NC,孵育72小时后,则与9-NC相当,甚至强于9-NC,经脂质体纳米化的9-NC并没有降低它的药理作用,能显著上调Bax/Bcl-2,还具有明显的缓释作用,9-NC-LP是一种具有可观的应用前景的抗肿瘤药物。

关键词： 9-硝基喜树碱脂质体细胞周期细胞凋亡 tfk-1

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