目的将表达CXCR4及增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)-CXCR4质粒转入肾癌细胞A498中,并建立稳定转染细胞株。方法针对CXCR4基因构建稳定表达CXCR4质粒,应用脂质体转染技术将稳定表达CXCR4及EGFP-CXCR4质粒...目的将表达CXCR4及增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)-CXCR4质粒转入肾癌细胞A498中,并建立稳定转染细胞株。方法针对CXCR4基因构建稳定表达CXCR4质粒,应用脂质体转染技术将稳定表达CXCR4及EGFP-CXCR4质粒转入肾癌A498细胞中,经过G418抗性筛选细胞株。通过共聚焦显微镜观察转染EGFP-CXCR4质粒的A498细胞的表达情况。通过共聚焦显微镜观察EGFP-CXCR4融合蛋白在A498经SDF-1刺激前后的变换。通过蛋白质印迹法检测转染后CXCR4蛋白表达水平的变化,应用MTT法检测转染后的A498细胞增殖能力水平,并通过Transwell实验观察稳定表达CXCR4的肾癌A498细胞株侵袭能力的改变。结果稳定表达CXCR4及EGFP-CXCR4的质粒构建后测序结果与CXCR4DNA序列完全吻合。质粒转入A498细胞后进行G418抗性筛选挑选出合适的细胞株。共聚焦显微镜观察发现,转染EGFP-CXCR4质粒的A498细胞中胞膜及胞质中均有绿色荧光表达,经SDF-1刺激后EGFP-CXCR4向细胞内转移。蛋白质印迹法发现稳定转染CXCR4质粒的A498细胞的CXCR4表达水平高于正常A498细胞。第3天以后,转染pcDNA-CXCR4及pEGFP-CXCR4质粒组的A498细胞增殖水平率高于正常A498细胞(P<0.01)。Transwell实验证实稳定表达CXCR4的肾癌A498细胞株侵袭能力与正常A498细胞株相比增强(P<0.01)。结论成功地构建了CXCR4稳定表达的肾癌A498细胞株,转染后A498细胞的增殖能力增强,侵袭能力增强,为后续实验奠定了基础。展开更多
Objective of the study:We used fluorescence imaging methods of apoptosis and necrosis in human renal carcinoma A498 tumor cells in vitro to reveal the indicated forms of cell death under the combined effect of flavono...Objective of the study:We used fluorescence imaging methods of apoptosis and necrosis in human renal carcinoma A498 tumor cells in vitro to reveal the indicated forms of cell death under the combined effect of flavonoid-containing extract of Gratiola officinalis and cytostatic(cyclo-phosphamide).Materials and methods:The dyes were propidium iodide and acridine orange,which were used in the"alive and dead"test.This test helped us to identify the total number of dead cells in the forms of necrosis and apoptosis and the number of cells in which apoptosis had started,it was characterized by the appearance of apoptotic bodies or nucleus pyknosis.Results:We found the most pronounced cytotoxic activity at the ratio of extract of Gratiola officinalis and cyclophosphamide concentrations of 1∶1.The number of living cells decreased when exposed to the ratio of extract and cytostatic concentrations of 2∶1.When the ratio of concentration of the extract relative to the cytostatic increased to 3∶1,the cytostatic activity of the extract began to appear,the total number of tumor cells decreased.The number of cells with nucleus pyknosis and the number of cells with apoptosis signs significantly increased at a 3∶1 ratio of extract and cytostatic concentrations,which confirms the presence of pro-apoptotic activity of the studied combination.This trend indicates the dependence of a certain form of cell death(apoptosis,necrosis)on the ratio of extract and cytostatic doses,and it also demon-strates the cytostatic and cytotoxic effects of this combination.Conclusion:Fluorescence methods of investigation in the"alive and dead"test allowed us to visualize the forms of cell death of human kidney carcinoma A498 by combined exposure to the fiavonoid-containing extract of Gratiola officinalis and cytostatic(cyclophosphamide)24 h after exposure.We found that the combination with a concentration ratio of the extract and cyclophosphamide of 3:1 has the greatest effectiveness due to stimulation of the cytostatic effect and cytotoxic effect.展开更多
文摘目的将表达CXCR4及增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)-CXCR4质粒转入肾癌细胞A498中,并建立稳定转染细胞株。方法针对CXCR4基因构建稳定表达CXCR4质粒,应用脂质体转染技术将稳定表达CXCR4及EGFP-CXCR4质粒转入肾癌A498细胞中,经过G418抗性筛选细胞株。通过共聚焦显微镜观察转染EGFP-CXCR4质粒的A498细胞的表达情况。通过共聚焦显微镜观察EGFP-CXCR4融合蛋白在A498经SDF-1刺激前后的变换。通过蛋白质印迹法检测转染后CXCR4蛋白表达水平的变化,应用MTT法检测转染后的A498细胞增殖能力水平,并通过Transwell实验观察稳定表达CXCR4的肾癌A498细胞株侵袭能力的改变。结果稳定表达CXCR4及EGFP-CXCR4的质粒构建后测序结果与CXCR4DNA序列完全吻合。质粒转入A498细胞后进行G418抗性筛选挑选出合适的细胞株。共聚焦显微镜观察发现,转染EGFP-CXCR4质粒的A498细胞中胞膜及胞质中均有绿色荧光表达,经SDF-1刺激后EGFP-CXCR4向细胞内转移。蛋白质印迹法发现稳定转染CXCR4质粒的A498细胞的CXCR4表达水平高于正常A498细胞。第3天以后,转染pcDNA-CXCR4及pEGFP-CXCR4质粒组的A498细胞增殖水平率高于正常A498细胞(P<0.01)。Transwell实验证实稳定表达CXCR4的肾癌A498细胞株侵袭能力与正常A498细胞株相比增强(P<0.01)。结论成功地构建了CXCR4稳定表达的肾癌A498细胞株,转染后A498细胞的增殖能力增强,侵袭能力增强,为后续实验奠定了基础。
基金The work was supported by State Assignment No.121032600197-2 of the Health Ministry of Russia.
文摘Objective of the study:We used fluorescence imaging methods of apoptosis and necrosis in human renal carcinoma A498 tumor cells in vitro to reveal the indicated forms of cell death under the combined effect of flavonoid-containing extract of Gratiola officinalis and cytostatic(cyclo-phosphamide).Materials and methods:The dyes were propidium iodide and acridine orange,which were used in the"alive and dead"test.This test helped us to identify the total number of dead cells in the forms of necrosis and apoptosis and the number of cells in which apoptosis had started,it was characterized by the appearance of apoptotic bodies or nucleus pyknosis.Results:We found the most pronounced cytotoxic activity at the ratio of extract of Gratiola officinalis and cyclophosphamide concentrations of 1∶1.The number of living cells decreased when exposed to the ratio of extract and cytostatic concentrations of 2∶1.When the ratio of concentration of the extract relative to the cytostatic increased to 3∶1,the cytostatic activity of the extract began to appear,the total number of tumor cells decreased.The number of cells with nucleus pyknosis and the number of cells with apoptosis signs significantly increased at a 3∶1 ratio of extract and cytostatic concentrations,which confirms the presence of pro-apoptotic activity of the studied combination.This trend indicates the dependence of a certain form of cell death(apoptosis,necrosis)on the ratio of extract and cytostatic doses,and it also demon-strates the cytostatic and cytotoxic effects of this combination.Conclusion:Fluorescence methods of investigation in the"alive and dead"test allowed us to visualize the forms of cell death of human kidney carcinoma A498 by combined exposure to the fiavonoid-containing extract of Gratiola officinalis and cytostatic(cyclophosphamide)24 h after exposure.We found that the combination with a concentration ratio of the extract and cyclophosphamide of 3:1 has the greatest effectiveness due to stimulation of the cytostatic effect and cytotoxic effect.
