| 摘要: |
| 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CXCR4 DNA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。 |
| 关键词: 肾肿瘤 A498细胞CXCR4受体 绿色荧光蛋白质类 转染 |
| DOI：10.3724/SP.J.1008.2013.00623 |
| 投稿时间：2013-02-03修订日期：2013-03-28 |
| 基金项目:国家自然科学基金(81272817/H1619). |
|
| Establishment of two renal carcinoma A498 cell lines stably expressing CXCR4 |
| WANG Lin-hui1△,WANG Zhi-xiang1,2△,LIU Bing1 ,YANG Qing1,SUN Ying-hao1* |
(1. Department of Urology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China
2. Department of Urology, No.188 Hospital of PLA, Chaozhou 521000, Guangdong, China
△Co-first authors.
*Corresponding author.) |
| Abstract: |
| Objective To transfect CXCR4 and enhanced green fluorescent protein (EGFP)-CXCR4 plasmids into renal carcinoma cell line A498 cells to prepare cell lines stably expressing CXCR4. Methods Two specific plasmids containing CXCR4 or EGFP-CXCR4 were transfected into renal cell carcinoma cell line A498. Then the cells stably expressing CXCR4 were screened by using G418. Confocal microscopy was used to observe the changes of EGFP-CXCR4 fusion protein in A498 cells before and after stimulation with SDF-1. Western blotting analysis was used to determine CXCR4 expression after transfection. Proliferation of A498 cells was detected by MTT and the invasion ability of cells was detected by transwell assay. Results The sequencing result of two plasmids was consistent with CXCR4 DNA sequence, and two cell lines were screened out by G418 screening after the plasmids were transfected into A498 cells. EGFP-CXCR4 fusion protein was found in the cell membrane and cytoplasm of EGFP-CXCR4 transfection group under confocal microscopy. EGFP-CXCR4 migrated into cells after SDF-1 stimulation. Western blotting analysis revealed higher CXCR4 expression in A498 cells stably transfected with CXCR4 plasmids compared with normal A498 cells. The proliferation of cells in pCNDA-CXCR4 and pEGFP-CXCR4 groups were significantly higher than that in normal A498 cell group (P<0.01). Transwell assay showed that the cell invasion ability of cells with stable CXCR4 expression was significantly increased compared with that in the normal A498 cell group (P<0.01). Conclusion We have successfully established A498 cell lines stably expressing CXCR4, which have enhanced proliferation levels and higher invasive ability. |
| Key words: kidney neoplasms A498 cells CXCR4 receptors green fluorescent proteins transfection |
