Objective: The aim of the study was to explore the effect of demethylating agent 5-Aza-2'-deoxycytidine (5-ADC) on expression of Fanconi anemia complementation group F (FANCF) gene and the proliferation of cervica...Objective: The aim of the study was to explore the effect of demethylating agent 5-Aza-2'-deoxycytidine (5-ADC) on expression of Fanconi anemia complementation group F (FANCF) gene and the proliferation of cervical cancer cells, to observe cell's sensitivity to chemotherapeutic drug taxol, and to explore the antitumor effect of 5-ADC as well as the new treatment of cervical cancer. Methods: Cervical cancer cell lines SiHa (FANCF gene full-methylated) and Hela (unmethylated) were treated with 5-ADC. We used the methylation-specific PCR (MSP), reverse transcription-polymerase chain reaction (RT-PCR) and Western blot to detect the FANCF methylation, mRNA and protein respectively. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect the proliferation of cells. The cytotoxicity of taxol was measured by flow cytometer. The nude mice bearing SiHa was used to observe the effect of 5-ADC in vivo. Results: Inhibition of DNA promoter methylation by 5-ADC reactivated the expression of FANCF mRNA and protein in SiHa cells, consistent with decreased growth speed and increased taxol resistance. These results were proven in experiments in vivo. Conclusion: The 5-ADC probably become a potential treatment drug through inhibiting the proliferation of cervical cancer cells in taxol-resistant patients.展开更多
以‘伯克利’蓝莓花芽为材料,研究了其在内休眠及解除休眠过程中部分生理生化指标的变化,并对内休眠与解除休眠花芽进行了基因组DNA甲基化敏感扩增多态性(methylation sensitive amplifi ed polymorphism,MSAP)分析。结果显示,蓝莓花芽...以‘伯克利’蓝莓花芽为材料,研究了其在内休眠及解除休眠过程中部分生理生化指标的变化,并对内休眠与解除休眠花芽进行了基因组DNA甲基化敏感扩增多态性(methylation sensitive amplifi ed polymorphism,MSAP)分析。结果显示,蓝莓花芽中脯氨酸、可溶性糖、可溶性蛋白含量及SOD、POD活性的变化趋势与休眠进程密切相关。内休眠期间,脯氨酸含量呈先降后升的趋势,可溶性糖含量及POD活性持续上升,可溶性蛋白含量及SOD活性持续下降;随着休眠开始解除,可溶性糖、可溶性蛋白含量及SOD活性均上升,而POD活性下降。MSAP分析表明,蓝莓花芽内休眠期间,DNA甲基化水平较高,并以全甲基化为主;内休眠花芽总甲基化率(38.41%)、全甲基化率(27.09%)及半甲基化率(11.32%)均明显高于解除休眠花芽;相对于内休眠花芽,解除休眠花芽约84.48%的位点发生了甲基化模式变化,其中去甲基化位点数和比例分别为248和40.52%,超甲基化的位点数和比例分别为185和30.23%。DNA甲基化参与了蓝莓内休眠的调控。展开更多
目的：建立孕期苯巴比妥暴露所致的IUGR模型,观察胎肾上腺胆固醇供应关键基因——清道夫受体B类I型（scavenger receptor class B type I,SR-BI）与乙酰乙酰辅酶A合成酶（acetoacetyl-CoA synthetase,AACS）的表达与DNA甲基化修饰改变,...目的：建立孕期苯巴比妥暴露所致的IUGR模型,观察胎肾上腺胆固醇供应关键基因——清道夫受体B类I型（scavenger receptor class B type I,SR-BI）与乙酰乙酰辅酶A合成酶（acetoacetyl-CoA synthetase,AACS）的表达与DNA甲基化修饰改变,验证此关键基因的DNA高甲基化作为胎肾上腺化合物发育毒性评价标志物的可能性。方法：健康Wistar孕鼠于受孕第7~17天灌胃给予50 mg·kg-1·d-1苯巴比妥或等量生理盐水,孕第17天处死,记录胎鼠体质量与胎盘重量,计算宫内发育迟缓（intrauterine growth retardation,IUGR）发生率。收集胎肾上腺用于透射电镜、荧光实时定量反转录PCR（reverse-transcription PCR,RTPCR）及亚硫酸氢盐测序PCR（bisulfite sequencing PCR,BSP）检测。结果：与对照组相比,苯巴比妥暴露组胎鼠体质量与胎盘重量均降低（P〈0.01）,IUGR发生率升高（P〈0.01）;透射电镜结果显示,胎肾上腺皮质细胞表现出线粒体水肿、胞浆内空泡等现象;RT-PCR检测结果显示,SR-BI与AACS表达降低（P〈0.05,P〈0.01）;BSP结果显示,SR-BI与AACS近端启动子区DNA甲基化频率升高（P〈0.01）。结论：孕期苯巴比妥暴露所致IUGR模型中,胎肾上腺组织结构受损,胆固醇供应关键基因SR-BI与AACS的mRNA表达改变,SR-BI与AACS的DNA高甲基化可作为潜在的化合物发育毒性评价标志物。展开更多
基金Supported by the grant from the National Science Foundation of Chongqing (No. cstc2011jjA10081)
文摘Objective: The aim of the study was to explore the effect of demethylating agent 5-Aza-2'-deoxycytidine (5-ADC) on expression of Fanconi anemia complementation group F (FANCF) gene and the proliferation of cervical cancer cells, to observe cell's sensitivity to chemotherapeutic drug taxol, and to explore the antitumor effect of 5-ADC as well as the new treatment of cervical cancer. Methods: Cervical cancer cell lines SiHa (FANCF gene full-methylated) and Hela (unmethylated) were treated with 5-ADC. We used the methylation-specific PCR (MSP), reverse transcription-polymerase chain reaction (RT-PCR) and Western blot to detect the FANCF methylation, mRNA and protein respectively. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect the proliferation of cells. The cytotoxicity of taxol was measured by flow cytometer. The nude mice bearing SiHa was used to observe the effect of 5-ADC in vivo. Results: Inhibition of DNA promoter methylation by 5-ADC reactivated the expression of FANCF mRNA and protein in SiHa cells, consistent with decreased growth speed and increased taxol resistance. These results were proven in experiments in vivo. Conclusion: The 5-ADC probably become a potential treatment drug through inhibiting the proliferation of cervical cancer cells in taxol-resistant patients.
文摘以‘伯克利’蓝莓花芽为材料,研究了其在内休眠及解除休眠过程中部分生理生化指标的变化,并对内休眠与解除休眠花芽进行了基因组DNA甲基化敏感扩增多态性(methylation sensitive amplifi ed polymorphism,MSAP)分析。结果显示,蓝莓花芽中脯氨酸、可溶性糖、可溶性蛋白含量及SOD、POD活性的变化趋势与休眠进程密切相关。内休眠期间,脯氨酸含量呈先降后升的趋势,可溶性糖含量及POD活性持续上升,可溶性蛋白含量及SOD活性持续下降;随着休眠开始解除,可溶性糖、可溶性蛋白含量及SOD活性均上升,而POD活性下降。MSAP分析表明,蓝莓花芽内休眠期间,DNA甲基化水平较高,并以全甲基化为主;内休眠花芽总甲基化率(38.41%)、全甲基化率(27.09%)及半甲基化率(11.32%)均明显高于解除休眠花芽;相对于内休眠花芽,解除休眠花芽约84.48%的位点发生了甲基化模式变化,其中去甲基化位点数和比例分别为248和40.52%,超甲基化的位点数和比例分别为185和30.23%。DNA甲基化参与了蓝莓内休眠的调控。
文摘目的：建立孕期苯巴比妥暴露所致的IUGR模型,观察胎肾上腺胆固醇供应关键基因——清道夫受体B类I型（scavenger receptor class B type I,SR-BI）与乙酰乙酰辅酶A合成酶（acetoacetyl-CoA synthetase,AACS）的表达与DNA甲基化修饰改变,验证此关键基因的DNA高甲基化作为胎肾上腺化合物发育毒性评价标志物的可能性。方法：健康Wistar孕鼠于受孕第7~17天灌胃给予50 mg·kg-1·d-1苯巴比妥或等量生理盐水,孕第17天处死,记录胎鼠体质量与胎盘重量,计算宫内发育迟缓（intrauterine growth retardation,IUGR）发生率。收集胎肾上腺用于透射电镜、荧光实时定量反转录PCR（reverse-transcription PCR,RTPCR）及亚硫酸氢盐测序PCR（bisulfite sequencing PCR,BSP）检测。结果：与对照组相比,苯巴比妥暴露组胎鼠体质量与胎盘重量均降低（P〈0.01）,IUGR发生率升高（P〈0.01）;透射电镜结果显示,胎肾上腺皮质细胞表现出线粒体水肿、胞浆内空泡等现象;RT-PCR检测结果显示,SR-BI与AACS表达降低（P〈0.05,P〈0.01）;BSP结果显示,SR-BI与AACS近端启动子区DNA甲基化频率升高（P〈0.01）。结论：孕期苯巴比妥暴露所致IUGR模型中,胎肾上腺组织结构受损,胆固醇供应关键基因SR-BI与AACS的mRNA表达改变,SR-BI与AACS的DNA高甲基化可作为潜在的化合物发育毒性评价标志物。
