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目的:构建含有小鼠CXCR3基因的重组逆转录病毒载体,获得稳定表达小鼠CXCR3分子的基因转染细胞株L929-mCXCR3,研究CXCR3与其配IP-10相互作用引起的L929-mCXCR3的迁移效应。方法:取1只雌性BALB/c小鼠(7周龄),尾静脉注射0.5mgConA/只,12h后取其脾脏,研磨成细胞悬液后,分离获得脾脏细胞;用含5万U/L人IL-2的RPMI1640培养基培养3d;收集细胞,TRIzol一步法抽提总RNA,RT-PCR扩增小鼠CXCR3全长基因,装入逆转录病毒载体pEGZ-term,与两辅助病毒载体脂质体法共转染包装细胞293T,收集含完整逆转录病毒颗粒的293T培养上清感染L929细胞,重复感染3次,筛选获得含Zeocin抗性的稳定表达小鼠CXCR3分子的基因转染细胞株。采用流式细胞术(FCM)和RT-PCR对CXCR3分子的表达进行鉴定。Transwell分析基因转染细胞株L929-mCXCR3在IP-10作用下的迁移能力。结果:构建了含小鼠CXCR3基因的重组逆转录病毒载体,建立了稳定表达小鼠CXCR3分子的基因转染细胞株L929-mCXCR3,其膜表面CXCR3分子阳性表达率为97.0%;该基因转染细胞株在IP-10的介导下可定向迁移,迁移率为4.356%。结论:L929-mCXCR3细胞株为研究CXCR3信号通路的生物学特性、肿瘤转移模型的建立和抗小鼠CXCR3单克隆抗体的研制奠定了基础。
OBJECTIVE: To construct a recombinant retrovirus vector containing mouse CXCR3 gene and obtain the gene transfection cell line L929-mCXCR3 stably expressing mouse CXCR3 molecule to study the migration effect of L929-mCXCR3 induced by CXCR3 and its interaction with IP-10. Methods: One female BALB / c mouse (7 weeks old) was injected with 0.5mgConA per mouse intravenously and the spleen was taken after 12h. The spleen cells were ground into cell suspension and isolated. Human IL-2 was cultured in RPMI1640 medium for 3 days. Cells were harvested and total RNA was extracted by TRIzol in one step. The full-length cDNA of mouse CXCR3 was amplified by RT-PCR and inserted into retroviral vector pEGZ-term. 293T cells were transfected with 293T recombinant plasmids and transfected into 293T culture supernatant containing intact retrovirus particles. The infected cells were infected three times, and the gene transfection cell line stably expressing CXCR3 gene containing Zeocin was obtained by screening. . The expression of CXCR3 molecules was identified by flow cytometry (FCM) and RT-PCR. Transwell analysis of gene transfected cell line L929-mCXCR3 in IP-10 migration. Results: The recombinant retroviral vector containing mouse CXCR3 gene was constructed and the gene transfection cell line L929-mCXCR3 stably expressing mouse CXCR3 gene was established. The positive rate of CXCR3 on the membrane surface was 97.0% Cell lines can be oriented migration under the IP-10-mediated mobility of 4.356%. Conclusion: The L929-mCXCR3 cell line lays the foundation for the study of the biological characteristics of CXCR3 signaling pathway, the establishment of tumor metastasis model and the development of anti-mouse CXCR3 monoclonal antibody.