目的利用RNA干扰稳定筛选-抑制技术,抑制人端粒酶逆转录酶(hTERT)基因表达,探讨靶向hTERT基因RNAi对肝癌细胞增殖的抑制效应。方法设计靶向hTERT基因的小干扰RNA,构建重组表达质粒pGenesil-shRNA-hTERT并导入人肝癌细胞系QGY细胞株,经G...目的利用RNA干扰稳定筛选-抑制技术,抑制人端粒酶逆转录酶(hTERT)基因表达,探讨靶向hTERT基因RNAi对肝癌细胞增殖的抑制效应。方法设计靶向hTERT基因的小干扰RNA,构建重组表达质粒pGenesil-shRNA-hTERT并导入人肝癌细胞系QGY细胞株,经G418筛选,建立稳定表达siRNA-hTERT的细胞株。采用real time RT-PCR、MTT和PCR-TRAP法同时检测pGenesil-shRNA-hTERT稳定抑制组和未处理QGY细胞组hTERT基因表达、端粒酶活性及细胞增殖变化。结果在稳定表达pGenesil-shRNA-hTERT的QGY细胞株中,RNAi效力持续、稳定存在,hTERT的mRNA表达、端粒酶活性明显降低,瘤细胞增殖被抑制。结论RNA干扰能持续、稳定地抑制靶基因hTERT的mRNA表达及肿瘤细胞增殖,是潜在的肿瘤基因治疗新方法。展开更多
目的:探讨乙型肝炎病毒X蛋白(hepatitis B virus X protein,HBx)参与人肝癌细胞hsp90α基因表达的调控机制。方法:采用半定量RT-PCR、蛋白质印迹法及荧光素酶活性检测技术,检测不同剂量HBx表达载体转染的HepG2细胞中hsp90α基因mRNA和...目的:探讨乙型肝炎病毒X蛋白(hepatitis B virus X protein,HBx)参与人肝癌细胞hsp90α基因表达的调控机制。方法:采用半定量RT-PCR、蛋白质印迹法及荧光素酶活性检测技术,检测不同剂量HBx表达载体转染的HepG2细胞中hsp90α基因mRNA和蛋白表达及启动子活性的变化,并观察转录因子c-Myc阻断剂10058-F4对上述效应的影响。采用启动子实验分析c-Myc对hsp90α基因启动子的激活作用。RT-PCR和蛋白质印迹法检测表达HBx的HepG2细胞中c-Myc mRNA和蛋白表达情况;蛋白质印迹法及ELISA分析细胞外信号调节激酶1/2(ERK1/2)和磷酸化ERK1/2(p-ERK1/2)表达水平及内源性ERK的活性。结果:HBx基因转染HepG2细胞中hsp90α基因的mRNA和蛋白表达水平以及启动子活性呈剂量-依赖性增强,P=0.006 1;c-Myc阻断剂抑制了HBx对hsp90α基因表达的上调效应。c-Myc通过与c-Myc位点(-1094/-1088)结合转录激活hsp90α基因启动子活性。HBx使HepG2细胞中c-Myc mRNA和蛋白表达增强,并伴有ERK1/2蛋白磷酸化水平增加,ERK活性明显提高,P=0.032。结论:HBx通过ERK信号通路反式激活肝癌细胞c-Myc介导的hsp90α基因启动子活性,从而上调hsp90α基因表达。展开更多
目的:研究赶黄草中木脂素类及黄酮类成分体外抑制肝癌细胞增殖的作用。方法:采用MTT法评价赶黄草中8个木脂素(1~8)和5个黄酮(9~13)体外抗人肝癌细胞SMMC-7721和Hep G2增殖的活性以及对正常肝细胞LO_2活力的影响。运用Image-i T DEAD Gre...目的:研究赶黄草中木脂素类及黄酮类成分体外抑制肝癌细胞增殖的作用。方法:采用MTT法评价赶黄草中8个木脂素(1~8)和5个黄酮(9~13)体外抗人肝癌细胞SMMC-7721和Hep G2增殖的活性以及对正常肝细胞LO_2活力的影响。运用Image-i T DEAD Green/Hoechst 33342荧光双染和高内涵成像分析化合物1对SMMC-7721细胞的影响。结果:化合物1、6、8~11可抑制SMMC-7721的增殖,6、9、11、13可抑制Hep G2的增殖。除化合物11以外,以上化合物对正常肝细胞LO_2均无明显细胞毒作用,且化合物3、4、5、13还可促进LO_2增殖。结论:赶黄草中部分木脂素及黄酮类成分不仅具有抑制肝癌细胞增殖的作用,而且对正常肝细胞无毒作用,甚至可保护正常肝细胞,表现出较好的选择性。展开更多
In this paper, the relationship between radiosensitivity, cell cycle alteration and the change of apoptosis in different human hepatoma cell lines irradiated by heavy ions were studied with the aim of building up the ...In this paper, the relationship between radiosensitivity, cell cycle alteration and the change of apoptosis in different human hepatoma cell lines irradiated by heavy ions were studied with the aim of building up the base data for clinical therapy. Exponentially growing hepatoma cell lines were irradiated by 80.55 MeV/u12C6＋ ions at a dose of 0 Gy, 0.5 Gy, 1 Gy, 2 Gy, 4 Gy and 8 Gy. The radiosensitivity was assessed by means of the colony-forming assay. The DNA content, the percentage of each cell-cycle phase and the apoptosis rate were obtained with flow cytometry methods. After the irradiation, the SF2 （survival fraction at 2 gray） of SMMC-7721 cells were evidently lower than that of HepG2 cells. The S phase arrest, G2/M phase arrest delay and the apoptosis in the two hepatoma cell lines varied with the increase of the dose and repair time. The heavy ions could obviously kill the human hepatoma cell lines. Compared to HepG2 cells, SMMC-7721 cells were more radiosensitive to 12C^6＋ ions.展开更多
文摘目的利用RNA干扰稳定筛选-抑制技术,抑制人端粒酶逆转录酶(hTERT)基因表达,探讨靶向hTERT基因RNAi对肝癌细胞增殖的抑制效应。方法设计靶向hTERT基因的小干扰RNA,构建重组表达质粒pGenesil-shRNA-hTERT并导入人肝癌细胞系QGY细胞株,经G418筛选,建立稳定表达siRNA-hTERT的细胞株。采用real time RT-PCR、MTT和PCR-TRAP法同时检测pGenesil-shRNA-hTERT稳定抑制组和未处理QGY细胞组hTERT基因表达、端粒酶活性及细胞增殖变化。结果在稳定表达pGenesil-shRNA-hTERT的QGY细胞株中,RNAi效力持续、稳定存在,hTERT的mRNA表达、端粒酶活性明显降低,瘤细胞增殖被抑制。结论RNA干扰能持续、稳定地抑制靶基因hTERT的mRNA表达及肿瘤细胞增殖,是潜在的肿瘤基因治疗新方法。
文摘目的:探讨乙型肝炎病毒X蛋白(hepatitis B virus X protein,HBx)参与人肝癌细胞hsp90α基因表达的调控机制。方法:采用半定量RT-PCR、蛋白质印迹法及荧光素酶活性检测技术,检测不同剂量HBx表达载体转染的HepG2细胞中hsp90α基因mRNA和蛋白表达及启动子活性的变化,并观察转录因子c-Myc阻断剂10058-F4对上述效应的影响。采用启动子实验分析c-Myc对hsp90α基因启动子的激活作用。RT-PCR和蛋白质印迹法检测表达HBx的HepG2细胞中c-Myc mRNA和蛋白表达情况;蛋白质印迹法及ELISA分析细胞外信号调节激酶1/2(ERK1/2)和磷酸化ERK1/2(p-ERK1/2)表达水平及内源性ERK的活性。结果:HBx基因转染HepG2细胞中hsp90α基因的mRNA和蛋白表达水平以及启动子活性呈剂量-依赖性增强,P=0.006 1;c-Myc阻断剂抑制了HBx对hsp90α基因表达的上调效应。c-Myc通过与c-Myc位点(-1094/-1088)结合转录激活hsp90α基因启动子活性。HBx使HepG2细胞中c-Myc mRNA和蛋白表达增强,并伴有ERK1/2蛋白磷酸化水平增加,ERK活性明显提高,P=0.032。结论:HBx通过ERK信号通路反式激活肝癌细胞c-Myc介导的hsp90α基因启动子活性,从而上调hsp90α基因表达。
文摘目的:研究赶黄草中木脂素类及黄酮类成分体外抑制肝癌细胞增殖的作用。方法:采用MTT法评价赶黄草中8个木脂素(1~8)和5个黄酮(9~13)体外抗人肝癌细胞SMMC-7721和Hep G2增殖的活性以及对正常肝细胞LO_2活力的影响。运用Image-i T DEAD Green/Hoechst 33342荧光双染和高内涵成像分析化合物1对SMMC-7721细胞的影响。结果:化合物1、6、8~11可抑制SMMC-7721的增殖,6、9、11、13可抑制Hep G2的增殖。除化合物11以外,以上化合物对正常肝细胞LO_2均无明显细胞毒作用,且化合物3、4、5、13还可促进LO_2增殖。结论:赶黄草中部分木脂素及黄酮类成分不仅具有抑制肝癌细胞增殖的作用,而且对正常肝细胞无毒作用,甚至可保护正常肝细胞,表现出较好的选择性。
基金the Ministry of Science and Technology,China(No.2003CCB00200)National Natural Science Foundation of China(No.10335050)
文摘In this paper, the relationship between radiosensitivity, cell cycle alteration and the change of apoptosis in different human hepatoma cell lines irradiated by heavy ions were studied with the aim of building up the base data for clinical therapy. Exponentially growing hepatoma cell lines were irradiated by 80.55 MeV/u12C6＋ ions at a dose of 0 Gy, 0.5 Gy, 1 Gy, 2 Gy, 4 Gy and 8 Gy. The radiosensitivity was assessed by means of the colony-forming assay. The DNA content, the percentage of each cell-cycle phase and the apoptosis rate were obtained with flow cytometry methods. After the irradiation, the SF2 （survival fraction at 2 gray） of SMMC-7721 cells were evidently lower than that of HepG2 cells. The S phase arrest, G2/M phase arrest delay and the apoptosis in the two hepatoma cell lines varied with the increase of the dose and repair time. The heavy ions could obviously kill the human hepatoma cell lines. Compared to HepG2 cells, SMMC-7721 cells were more radiosensitive to 12C^6＋ ions.
