莪术油诱导人胃癌SGC-7901细胞和鼻咽癌CNE细胞凋亡的研究
摘要
细胞凋亡是细胞死亡形式之一,它以细胞DNA发生特异性的降解,形态上表现为核固缩、胞膜发泡和凋亡小体形成为特征,是由特定的基因调控,无明显细胞溶解的自杀过程。诸多生理及病理过程均有凋亡参与调节。用荧光显微镜及透射电镜可观察到凋亡细胞呈现为核浓缩、核碎裂、核边聚、细胞皱缩、细胞以出穿的方式形成凋亡小体等典型的凋亡形态学特征,琼脂糖电泳示DNA呈梯带。近年来随着分子肿瘤学、分子药理学的不断发展,以及对肿瘤本质的阐明,大规模、快速筛选组合化学、基因工程等先进技术的发明和应用加速了药物开发进程。抗肿瘤药物的研究与开发已进入一个崭新的时代。
本研究以人胃癌SGC-7901细胞和鼻咽癌CNE细胞为研究对象,探讨莪术油诱导人胃癌SGC-7901细胞和鼻咽癌CNE细胞凋亡的作用。采用台盼蓝拒染法检测不同剂量莪术油对人胃癌SGC-7901细胞和鼻咽癌CNE细胞的增殖抑制作用。光学显微镜,荧光显微镜和透射电镜观察细胞的形态学变化,DNA琼脂糖凝胶电泳检测细胞DNA片段化情况,流式细胞术检测细胞线粒体膜电位的改变,凋亡率和细胞周期分布。结果显示莪术油作用人胃癌SGC-7901细胞和鼻咽癌CNE细胞凋亡48h均呈现不同程度的凋亡特征,且有量效关系。体外培养的人鼻咽癌CNE细胞对莪术油的敏感性高于人胃癌SGC-7901细胞。莪术油作用人胃癌SGC-7901细胞和鼻咽癌CNE细胞48h诱导凋亡的最佳浓度分别为2.25μg/mL,2.2μg/mL。
Apoptosis is one form of cell death, which occurred in cell-specific DNA degradation, the form for the performance of karyopyknosis, membrane and foam became apoptotic body features, from a specific gene regulation.There is no significant cells of suicide process of dissolution. Many physiological and pathological process are involved in regulating apoptosis.Fragmentation as well as formation of apoptotic bodies was observed by fluorescent stain, and transmission electron microscopy. Biochemically, DNA appeared as ladder in agarose gel electrophoresis.In recent years, as the molecular oncology, molecular pharmacology continues to develop, as well as to clarify the nature of the tumor, large-scale, rapid screening of combinatorial chemistry, genetic engineering and other advanced technology to accelerate the invention and application of the drug development process. Anticancer drug research and development has entered a new era.
To use the gastric cancer line SGC-7901 and Nasopharyngeal cancer cells line CNE for research, evaluating the anti-tumor effect of zedoary turmerie oil on the gastric cancer line SGC-7901 and Nasopharyngeal cancer cells line CNE and exits mechanism. Cell viability and proliferration assay was evaluated by trypan blue-rejected staining method.Cells are observed under light microscope,fluorescence microscope and electron transmission microscopy. The DNA fragments are also analyzed by agarose gel electrophoresis to testify apoptosis. The flow cytometry (FCM) is used to investigate the changes of mitochondrial membrene potential(DYm), the apoptotic rate and changes of cell cycle distribution.The results showed that the role of ZTO in SGC-7901 and CNE after all with varying degrees of apoptosis. And with time and dose of the drug increased significantly. Nasopharyngeal cancer cells line CNE is more sensitive than gastric cancer line SGC-7901 to ZTO in vitro.Through these experiments,it find that the concentrations which ZTO can induce SGC-7901 apoptosis is 2.25μg/mL in 48h, and CNE is 2.2μg/mL in 48h,48 hours is the best time.
本研究以人胃癌SGC-7901细胞和鼻咽癌CNE细胞为研究对象,探讨莪术油诱导人胃癌SGC-7901细胞和鼻咽癌CNE细胞凋亡的作用。采用台盼蓝拒染法检测不同剂量莪术油对人胃癌SGC-7901细胞和鼻咽癌CNE细胞的增殖抑制作用。光学显微镜,荧光显微镜和透射电镜观察细胞的形态学变化,DNA琼脂糖凝胶电泳检测细胞DNA片段化情况,流式细胞术检测细胞线粒体膜电位的改变,凋亡率和细胞周期分布。结果显示莪术油作用人胃癌SGC-7901细胞和鼻咽癌CNE细胞凋亡48h均呈现不同程度的凋亡特征,且有量效关系。体外培养的人鼻咽癌CNE细胞对莪术油的敏感性高于人胃癌SGC-7901细胞。莪术油作用人胃癌SGC-7901细胞和鼻咽癌CNE细胞48h诱导凋亡的最佳浓度分别为2.25μg/mL,2.2μg/mL。
Apoptosis is one form of cell death, which occurred in cell-specific DNA degradation, the form for the performance of karyopyknosis, membrane and foam became apoptotic body features, from a specific gene regulation.There is no significant cells of suicide process of dissolution. Many physiological and pathological process are involved in regulating apoptosis.Fragmentation as well as formation of apoptotic bodies was observed by fluorescent stain, and transmission electron microscopy. Biochemically, DNA appeared as ladder in agarose gel electrophoresis.In recent years, as the molecular oncology, molecular pharmacology continues to develop, as well as to clarify the nature of the tumor, large-scale, rapid screening of combinatorial chemistry, genetic engineering and other advanced technology to accelerate the invention and application of the drug development process. Anticancer drug research and development has entered a new era.
To use the gastric cancer line SGC-7901 and Nasopharyngeal cancer cells line CNE for research, evaluating the anti-tumor effect of zedoary turmerie oil on the gastric cancer line SGC-7901 and Nasopharyngeal cancer cells line CNE and exits mechanism. Cell viability and proliferration assay was evaluated by trypan blue-rejected staining method.Cells are observed under light microscope,fluorescence microscope and electron transmission microscopy. The DNA fragments are also analyzed by agarose gel electrophoresis to testify apoptosis. The flow cytometry (FCM) is used to investigate the changes of mitochondrial membrene potential(DYm), the apoptotic rate and changes of cell cycle distribution.The results showed that the role of ZTO in SGC-7901 and CNE after all with varying degrees of apoptosis. And with time and dose of the drug increased significantly. Nasopharyngeal cancer cells line CNE is more sensitive than gastric cancer line SGC-7901 to ZTO in vitro.Through these experiments,it find that the concentrations which ZTO can induce SGC-7901 apoptosis is 2.25μg/mL in 48h, and CNE is 2.2μg/mL in 48h,48 hours is the best time.
引文
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[3] Bayly AC, Roberts RA, Dive C. Suppression of liver cell apoptosis in vitro by the nongenotoxic hepatocarcinogen and peroxisome proliferator nafenopin. J Cell Bio,2004;125:197-203
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[6]曹玲,万嵘,王玉.反相高效液相色谱法测定莪术油注射液中莪术醇和牦牛儿酮的量[J].中国生化药物杂志,2007,28(6):394-396.
[7]宋爱莉,殷玉琨,莪术油千预治疗肿瘤的研究及应用概况[J].山东中医药大学学报,2008,32(2):172·174.
[8]黄艰东,项琪,姚崇舜,等.莪术油喷雾剂的研制及抗病毒作用的实验研究[J].中药材,2007,30(3):342-345.
[9]竺远,金凌芳.莪术油葡萄糖注射液治疗毛细支气管炎疗效观察[J].现代中西医结合杂志,2005,14(11):1452.
[10]游剑,李青坡,于英伟.应用HPl￡研究莪术油的药动学[J].中国药学杂志,2005,40(5):380-382.
[11]程剑华,刘伟胜,吴万银,等。重要莪术油肝动脉灌注治疗原发性肝癌的临床研究[J]。中医杂志,1999,40(1);25-26。
[12]程剑华,常刚,吴万银,等。莪术油和化疗药对照肝动脉灌注栓塞治疗原发性肝癌的临床研究[J]。中国中西医结合杂志,2001,21(3):165-167。
[13]徐凯,罗海英,李柳宁,等。中药灌注栓塞为主综合治疗原发性肝癌的临床研究[J]。中国中西医结合杂志,2005,25(4):299-302。
[14]吴万银,徐凯,刘伟胜,等。莪术油微球经肝动脉灌注栓塞治疗原发性肝癌[J]。上海中医药杂志,2004,38(4):3-6。
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