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目的:构建人CXCR4及CCR5真核表达重组质粒,转染人卵巢癌细胞SKOV3,建立稳定转染细胞系并观察其表达效果。方法:从人外周血单个核细胞中提取RNA,采用反转录PCR技术扩增CXCR4及CCR5的基因编码序列,将序列克隆至真核表达载体pEGFP,经酶切和测序鉴定后,应用脂质体转染技术将质粒cancer.pEGFP-CXCR4和pEGFP-CCR5分别导入不表达CXCR4及CCR5蛋白的SKOV3细胞,经G418抗性筛选得到阳性细胞克隆并扩大培养成系。分别采用免疫荧光染色和流式细胞术方法(FCM)检测稳定转染细胞株CXCR4和CCR5的表达。结果:构建了真核表达载体pEG-FP-CXCR4和pEGFP-CCR5;得到了抗G418阳性细胞克隆;免疫荧光染色和FCM检测结果显示,转染质粒的SKOV3细胞表达CXCR4和CCR5。结论:成功建立稳定表达趋化因子受体CXCR4和CCR5的卵巢癌细胞株,为CXCR4和CCR5在卵巢癌中的研究工作提供依据及平台。
OBJECTIVE: To construct human CXCR4 and CCR5 eukaryotic expression plasmids and transfect human ovarian cancer cell line SKOV3 to establish stable transfected cell lines and observe their expression. Methods: RNA was extracted from human peripheral blood mononuclear cells. The coding sequence of CXCR4 and CCR5 was amplified by reverse transcription PCR. The sequence was cloned into eukaryotic expression vector pEGFP. After digestion and sequencing, The plasmid cancer.pEGFP-CXCR4 and pEGFP-CCR5 were respectively transfected into SKOV3 cells that do not express CXCR4 and CCR5 proteins by using the transfection technique. The positive clones were screened by G418 resistance and expanded to culture. The expression of CXCR4 and CCR5 in stable transfected cells was detected by immunofluorescence staining and flow cytometry (FCM) respectively. Results: The eukaryotic expression vectors pEG-FP-CXCR4 and pEGFP-CCR5 were constructed. The anti-G418 positive cell clones were obtained. Immunofluorescence staining and FCM showed that CXCR4 and CCR5 were expressed in SKOV3 cells. CONCLUSION: Ovarian cancer cell lines stably expressing chemokine receptors CXCR4 and CCR5 have been successfully established and provide a basis and platform for the study of CXCR4 and CCR5 in ovarian cancer.