Construction Of Mouse CXCR3A And Human CXCR3B Gene Transfected Cell Lines, Identification And Study Of Their Biological Functions

CXC Chemokine Receptor 3——CXCR3(CD183)is a 7-time transmembrane G protein couple receptor. It is known that there are three splice variant, CXCR3-A (namely CXCR3), CXCR3-B and CXCR3-alt. CXCR3A mainly presents at activated and (or) memory T cell, NK cell and Macrophage cell surface. Its chemotatic factor ligands are CXCL9/Mig, CXCL10/IP-10 and CXCL11/I-TAC. The recent researchs show that CXCR3A is highly expressed on malignant tumor cell surface and mediates tumor transfer and invasion through receptor-ligand interaction. CXCR3-B mainly expressed in human microvascular endothelial cells. Besides with CXCL9/Mig, CXCL10/IP-10 and CXCL11/I-TAC, it also acts as functional receptor for CXCL4/PF4. The recent researchs show that CXCR3B could induce cell apoptosis through binding its ligands on the endothelial cells.Therefore, the aim of study is to clone mouse CXCR3A gene and establish gene transfection cell lines B16-mCXCR3A with stably expression of mouse CXCR3A molecule, and further investigates the role of the mouse gene CXCR3A in the process of tumor formation and metastasis. Moreover, cloning human CXCR3B gene and establishment gene transfection cell lines L929-huCXCR3B with stable expression of human CXCR3B molecule lay material foundation for establishment mouse anti human CXCR3B mAb.PartⅠMouse CXCR3A gene transfected cell line B16-mCXCR3A establishmentObjective:To clone mouse CXCR3A coding region gene and construct an engineered B16 cell line which constantly express the mouse CXCR3A gene.Methods:Mouse CXCR3A gene of full length was amplified by PCR from the plasmid pMD19-T/mCXCR3A. Then, it was inserted into eukaryotic expression vector pIRES2-EGFP to construct recombinant vector pIRES2-EGFP/mCXCR3A. The recombinant vector was transfected into B16 cells with Lipofectamine TM 2000,and cell clones stably expressing mouse CXCR3A molecule were screened by G418.We used FCM and RT-PCR to verify expression of CXCR3A from protein level and gene level.Results:We have constructed the eukaryotic transfer vector pIRES2-EGFP/mCXCR3A and screened for the engineered cell line B16-mCXCR3A that constantly express the mouse CXCR3A gene.Conclusion:We successfully constructed an engineered B16 cell line which constantly express the mouse CXCR3A gene.PartⅡStudy the migration and oncogenic function of B16-mCXCR3A cell line in vitro and in vivoObjective:To investigate the role of the mouse gene CXCR3A in the process of tumor formation and metastasis.Methods:Transwell system was used to explore the migration ability of B16-mCXCR3A cells under the condition of IP-10. B16-mCXCR3A cells were introduced into the BALB/c mouse by subcutaneous inoculation and eye intravenous injection respectively to investigate their oncogenic and migration capabilities.Results:The result that 3208 B16-mCXCR3A cells of the total 1x105 cells migrated under the induction of IP-10 showed statistically significant difference(P<0.01)compared with B16 cells and B16-mock treated cells. Subcutaneous inoculation experiments demonstrated that the tumor formation rate of B16-mCXCR3A, B16 and B16-mock treated cells all reached 100%. 20 days after inoculation, when intravenous injection with B16-mCXCR3A cells, about 50% mouses exhibited obvious black tumor metastases while these black tumor metastases were not observed in mouses inoculated with B16 and B16-mock treated cells. The result of B16-mCXCR3A treated cells showed statistically significant difference(P<0.01)compared to B16 cells and B16-mock treated cells.Conclusion:B16-mCXCR3A cells woule migrate under the meditation of IP-10 and could up-regulate the tumor metastasis rate . PartⅢConstruction of human CXCR3B gene transfected cell line, identification and study of its biological functionObjective:To clone human CXCR3B coding region gene and construct an engineered L929 cell line which constantly express the human CXCR3B gene.Methods:Human CXCR3B gene of full length was amplified by PCR from the plasmid pMD19-T/huCXCR3A. Then, it was inserted into eukaryotic expression vector pIRES2-EGFP to construct recombinant vector pIRES2-EGFP/huCXCR3B. The recombinant vector was transfected into L929 cells with Lipofectamine TM 2000,and cell clones stably expressing human CXCR3B molecule were screened by G418.We used FCM and RT-PCR to verify expression of CXCR3B from protein level and gene level. The ability of proliferation of L929-huCXCR3B under the circumstance of CXCR3B ligand called Mig was analyzed via MTT methods.Results:We have constructed recombinant human CXCR3B gene expression vector and obtained L929-huCXCR3B gene transfected cell line which can stably express human CXCR3B molecule. The positive expression rate of CXCR3B on L929-huCXCR3B cells was 93% .The result of MTT assay showed that the proliferation of L929-huCXCR3B cells were inhibited when the cells were cocultured with Mig after 24h,48h and 72h ,and the inhibition ratio were 41.44%,44.01% and 24.80 % respectively.Conclusion:Construction of L929-huCXCR3B cell line has laid a good foundation on research of the CXCR3B signal pathway and preparation of the CXCR3B monoclonal antibody.