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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。
Objective To transfer CXCR4 and enhanced green fluorescent protein (EGFP) -CXCR4 into renal cell line A498 and to establish a stable transfected cell line. Methods The CXCR4 plasmid was constructed stably expressing CXCR4 gene. The stable expression CXCR4 and EGFP-CXCR4 plasmids were transfected into renal cell carcinoma A498 cells by lipofection technique, and the cell lines were screened by G418 resistance. The expression of EGFP-CXCR4 plasmid-transfected A498 cells was observed by confocal microscopy. Confocal microscope was used to observe the change of EGFP-CXCR4 fusion protein before and after A498 stimulation by SDF-1. The expression of CXCR4 protein was detected by Western blotting. The proliferation of A498 cells was detected by MTT assay. The invasion ability of A498 cell line stably expressing CXCR4 was observed by Transwell assay. Results The constructed plasmids stably expressing CXCR4 and EGFP-CXCR4 were completely consistent with the CXCR4 DNA sequence. The plasmids were transfected into A498 cells and screened by G418 to select appropriate cell lines. Confocal microscopy showed EGFP-CXCR4 transfected EGFP-CXCR4 plasmid in the cytoplasm and cytoplasm were green fluorescent expression, after EGFP-CXCR4 SDF-1 transfected cells. Western blotting revealed that the CXCR4 expression level was higher in A498 cells stably transfected with the CXCR4 plasmid than in normal A498 cells. After day 3, the proliferation of A498 cells transfected with pcDNA-CXCR4 and pEGFP-CXCR4 plasmid groups was higher than that of normal A498 cells (P <0.01). The results of Transwell assay showed that the invasion ability of A498 cell line stably expressing CXCR4 was enhanced compared with that of A498 cell line (P <0.01). Conclusions A498 cell line stably expressing CXCR4 was constructed successfully. After transfection, A498 cells proliferated and the invasive ability was enhanced, which laid the foundation for subsequent experiments.