论文部分内容阅读
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20μg/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: To establish B16 cell line stably expressing mouse CXCR3 gene and study the role of CXCR3 in tumor formation and metastasis. Methods: CXCR3 gene was amplified from pMD19-T / mCXCR3 plasmid by PCR and inserted into eukaryotic expression vector pIRES2-EGFP. Lipofectamine was used to transfect mouse B16 melanoma cells. The positive clones were screened by G418, The expression of CXCR3 at mRNA and protein levels in positive clones was analyzed by RT-PCR and immunofluorescence techniques. Transwell system was used to detect the migration of B16-mCXCR3 cells under the ligand IP-10. The B16-mCXCR3 cells were inoculated into BALB / c mice by subcutaneous and ophthalmic injection, respectively. The rate of tumorigenesis and tumor metastasis in mice were observed. Results: The eukaryotic expression vector pIRES2-EGFP / mCXCR3 expressing mouse CXCR3 gene was constructed and transfected into B16-mCXCR3 cells stably expressing CXCR3. The number of migrating cells in B16-mCXCR3 cells (1 × 105 cells) was 3 208 after 20μg / L IP-10 treatment, which was statistically significant compared with B16 and B16-mock groups (P <0.01). B16-mCXCR3 cells, B16 cells and B16-mock cells were inoculated subcutaneously in mice, and the tumor formation rates on day 20 were both 100%. B16-mCXCR3 cells were injected intraocularly into the mice. On day 21, 50% of the mice showed macroscopic metastases of black tumors in the lungs. No tumor metastasis was seen in the lungs of the B16 and B16-mock cells, Compared with B16 group and B16-mock group, mCXCR3 group had statistical significance (P <0.01). CONCLUSION: B16-mCXCR3 cells transfected with CXCR3 gene can migrate in the direction of IP-10 and increase the metastasis rate in mice.