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
SDF-1α,a ligand for the chemokine receptor CXCR4,is well known for mediating the migration of breast cancer cells.In a previous study we demonstrated that a synthetic 21-mer peptide antagonist of CXCR4（NT21MP） deri...SDF-1α,a ligand for the chemokine receptor CXCR4,is well known for mediating the migration of breast cancer cells.In a previous study we demonstrated that a synthetic 21-mer peptide antagonist of CXCR4（NT21MP） derived from the viral macrophage inflammatory protein Ⅱ could antagonize tumor growth in vivo by inhibiting cellular proliferation and inducing apoptosis in breast cancer cells.However,the role of SDF-1α in the signaling pathways underlying the proliferation of human breast cancer cells and associated signaling pathways and inhibiting signal pathways of NT21MP remained unclear.The present study investigated the mechanism of NT21MP on anti-tumor in breast cancer in vitro.The effect of NT21MP on the viability of cells was determined by the MTT assay.Annexin V-FITC and PI staining was performed to detect early stage apoptosisin SKBR3 cells treated with SDF-1α and AMD3100 or NT21MP.Western blotting techniques were used to assay the composition of phosphoproteomics and total proteins present in the SKBR3 breast cancer cells.RT-PCR and Western blotting technique were used to detect the effect of NT21MP and AMD3100 on Bcl-2 and Bax expression.The results indicated that SDF-1α prevented apoptosis and promoted the proliferation of SKBR3 human breast cancer cells.As compared with untreated SKBR3 cells,Treatment with SDF-1α significantly increased cell viability,and NT21MP abolished the protective effects of SDF-1α dose-dependently（P0.05）.There was a significant decrease in the percentage of apoptotic cells after SDF-1α treatment as compared with control group（2.7%±0.2% vs.5.7%±0.4%,P0.05）.But pretreatment of SKBR3 cells with NT21MP significantly attenuated the antiapoptotic effects of SDF-1α as compared with SKBR3 cells without NT21MP pretreatment.The proliferative and anti-apoptotic effects of SDF-1α in SKBR3 cells were associated with an increase in AKT and ERK1/2 phosphorylation as well as a decrease in Bax expression and an increase in Bcl-2 expression.These changes in intracellular processes were blocked by NT21MP in a dose-dependent manner（P0.05）.In conclusion,NT21MP efficiently inhibits SDF-1α-induced proliferation and antiapoptosis in SKBR3 cells by reducing the levels of phosphorylated AKT and ERK1/2,as well as decreasing the ratio of expression of Bcl-2 relative to Bax.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supported by a grant from the National Natural Sciences Foundation of China (No. 81071848)a grant from a Key Program of Anhui Educational Committee(No. KJ2010A240)
文摘SDF-1α,a ligand for the chemokine receptor CXCR4,is well known for mediating the migration of breast cancer cells.In a previous study we demonstrated that a synthetic 21-mer peptide antagonist of CXCR4（NT21MP） derived from the viral macrophage inflammatory protein Ⅱ could antagonize tumor growth in vivo by inhibiting cellular proliferation and inducing apoptosis in breast cancer cells.However,the role of SDF-1α in the signaling pathways underlying the proliferation of human breast cancer cells and associated signaling pathways and inhibiting signal pathways of NT21MP remained unclear.The present study investigated the mechanism of NT21MP on anti-tumor in breast cancer in vitro.The effect of NT21MP on the viability of cells was determined by the MTT assay.Annexin V-FITC and PI staining was performed to detect early stage apoptosisin SKBR3 cells treated with SDF-1α and AMD3100 or NT21MP.Western blotting techniques were used to assay the composition of phosphoproteomics and total proteins present in the SKBR3 breast cancer cells.RT-PCR and Western blotting technique were used to detect the effect of NT21MP and AMD3100 on Bcl-2 and Bax expression.The results indicated that SDF-1α prevented apoptosis and promoted the proliferation of SKBR3 human breast cancer cells.As compared with untreated SKBR3 cells,Treatment with SDF-1α significantly increased cell viability,and NT21MP abolished the protective effects of SDF-1α dose-dependently（P0.05）.There was a significant decrease in the percentage of apoptotic cells after SDF-1α treatment as compared with control group（2.7%±0.2% vs.5.7%±0.4%,P0.05）.But pretreatment of SKBR3 cells with NT21MP significantly attenuated the antiapoptotic effects of SDF-1α as compared with SKBR3 cells without NT21MP pretreatment.The proliferative and anti-apoptotic effects of SDF-1α in SKBR3 cells were associated with an increase in AKT and ERK1/2 phosphorylation as well as a decrease in Bax expression and an increase in Bcl-2 expression.These changes in intracellular processes were blocked by NT21MP in a dose-dependent manner（P0.05）.In conclusion,NT21MP efficiently inhibits SDF-1α-induced proliferation and antiapoptosis in SKBR3 cells by reducing the levels of phosphorylated AKT and ERK1/2,as well as decreasing the ratio of expression of Bcl-2 relative to Bax.
