转染小鼠CXCR3基因的B16-mCXCR3细胞在体内外迁移和致瘤作用研究

摘要

目的:建立稳定表达小鼠CXCR3基因的B16细胞株,研究CXCR3分子在肿瘤形成及转移过程中的作用.方法:采用PCR方法从pMD19-T/mCXCR3质粒中扩增CXCR3基因,插入到真核表达载体pIRES2-EGFP中,脂质体法转染小鼠黑色素瘤细胞B16;G418加压筛选阳性克隆,分别用RT-PCR方法与免疫荧光技术分析阳性克隆中CXCR3在mRNA和蛋白水平的表达.采用Transwell系统检测B16-mCXCR3细胞在其配体IP-10介导下的迁移能力.将B16-mCXCR3细胞分别通过皮下和眼静脉注射接种于BALB/c小鼠,观察在小鼠体内的成瘤率及肿瘤转移情况.结果:构建了表达小鼠CXCR3基因的真核表达载体pIRES2-EGFP/mCXCR3,转染该载体后获得了稳定表达CXCR3的B16-mCXCR3细胞.B16-mCXCR3细胞(1×105个),在20 ìg/L IP-10作用下迁移的细胞数为3 208个,与B16组和B16-mock组相比均具有统计学意义(P＜0.01).于小鼠皮下接种B16-mCXCR3细胞、B16细胞和B16-mock细胞,第20天成瘤率均为100%.于小鼠眼静脉注射B16-mCXCR3细胞,第21天时50%的小鼠在肺部出现肉眼可见的黑色肿瘤转移灶,B16细胞组和B16-mock细胞组肺部未见肿瘤转移灶,B16-mCXCR3组与B16组和B16-mock组相比均具有统计学意义(P＜0.01).结论:转染CXCR3基因的B16-mCXCR3细胞,在IP-10介导下可定向迁移,并可增加在小鼠体内的转移率.%Objective: In order to investigate the role of mouse gene CXCR3 in the process of tumor formation and metastasis, to construct an engineered B16 cell line expressing the mouse CXCR3 gene constantly.Methods: The eukaryotic transfer vector pIRES2-EGFP-mCXCB3 used to transfect B16 cells was constructed by inserting the PCR-amplified CXCR3 cassette from the plasmid pMD19-T/mCXCR3 between the EcoR I and BamH I sites of the eukaryotic expression vector pIRES2-EGFP and then the transfer vector was transfected into B16 cells by the liposome-mediated methods.The positive B16-mCXCR3 clones were screened for G418-resistance and protein expression of CXCR3 gene was further examined through the RT-PCR and Immunofluorescence methods.Transwell system was used to explore the migration ability of B16-mCXCR3 cells under the condition of IP-l0.B16-mCXCR3 cells were introduced into BALB/c mice by subcutaneous inoculation and intravenous injection in eye respectively to investigate the oncogenic and lung migration capabilities.Results: The eukaryotic transfer vector pIRES2-EGFP-mCXCR3 was constructed and screened in the engineered cell line B16-mCXCR3 that constantly expressed the mouse CXCR3 gene.The resulting B16-mCXCR3 cells, with 3 208 cells out of the total 1 × 105 cells migrating under the induction of IP- 10,and showing statistically significant difference( P ＜ 0.01 ) compared to B16 cells and B16-mock-treuted cells.Subcutaneous inoculation experiments demonstrated that the tumor formation rate of B16-mCXCR3, B16 and Bl6-mock-treated cells all reached 100％ 20 days after inoculation, when intravenous injection with B16-mCXCR3 cells,about 50％ of the mice exhibited obvious black tumor metastases while these black tumor metastases were not observed in mice inoculated with B16 and B16-mock treated cells.The resulting B16-mCXCR3 cells were shown with statistically significant difference( P ＜ 0.01 ) compared to B16 cells and B16-mock treated cells.Conclusion: B16-mCXCR3 cells transfected with the CXCR3 gene exhibis migration under the meditation of IP-10 and could up-regulate the tumor metastasis rate.