[Construction of human CXCR3B gene transfected cell line, identification and study of its biological function].

AIM: To construct recombinant human CXCR3B gene expression vector and obtain L929-CXCR3B gene transfected cell line for stably expressing human CXCR3B. 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 24 h, 48 h and 72 h, 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.

[Mouse anti-human CXCR3 mAb preparation and its biological function].

AIM: Obtain hybridoma cell line with continuing expression of mouse anti-human CXCR3 mAb, investigate expression characteristics of human CXCR3 and how CXCR3 signal transduction function on L929-huCXCR3 and colon carcinoma cell lines transfer and growth. METHODS: Taking L929-huCXCR3 cell with high expression of human CXCR3 membrane molecule as immunogen to immunize BALB/c mouse, we fused immunized mouse spleen cell with myeloma cell Sp2/0 of its same germ line, then used L929-huCXCR3 as screening cell and empty vector transfected cell L929-mock as negative control. Obtained hybridoma cell line with continuing secretion of anti-human CXCR3 mAb through flow cytometry. We used Ig subclass type rapid qualitation indicator paper method and indirect immunofluorescence to identify obtained hybridoma cell line and mAb, indirect immunofluorescence to analyze CXCR3 expression on tumor cell surface, Transwell isolation cabin to assess effect on L929-huCXCR3 and colon carcinoma cell line Colo205, HCT116 and HT29 migration by mAb, MTT method to analyze how mAb function on colon carcinoma cell line Colo205 proliferation. RESULTS: Obtained a hybridoma cell line with continuing secretion of mouse anti-human CXCR3 mAb, named 9B5. According to rapid qualitation test paper analysis, light chain of the mAb was chain and heavy chain is IgG1 subclass. Indirect immunofluorescence and flow cytometry results show that the mAb can recognize CXCR3 molecules on the surface of activated T lymphocyte and colon carcinoma cell line Colo205, HCT116 and HT29 cell. mAb 9B5 can inhibit oriented migration of L929-huCXCR3 cells, colon carcinoma cell line Colo205, HCT116 and HT29 cell, it can also inhibit Colo205 growth promotion effect by IP-10. CONCLUSION: Successfully obtain a hybridoma cell line with continuing secretion of mouse anti-human CXCR3 mAb, which has laid material foundation on investigation of CXCR3 expression characteristics and CXCR3 signal transduction function on tumor growth and migration. It is prospective to create a new way and a new drug for treatment of tumor metastasis.

[Construction of murine CXCR3 gene transfected cell line, identifiction and study of its biological function].

AIM: To construct recombinant murine CXCR3 gene retroviral vector and obtain L929-mCXCR3 gene transfected cell line for stably expressing murine CXCR3. We further study on L929-mCXCR3 migration effect resulted from interaction by CXCR3 and its ligand IP-10. METHODS: One female BALB/c mouse (7 weeks) was injected with 0.5 mg Con A intravenously (i.v.) via a tail vein. Twelve hours later the mouse was sacrificed and the spleen was removed.The spleen was pressed through a 150 microm stainless steel mesh. The isolated splenocytes were cultured in RPMI1640 supplemented with 50 U/mL human IL-2 for 3 days.Total RNA was extracted with TRIzol. Murine CXCR3 gene of full length was amplified by RT-PCR, then, it was inserted into retrovirus vector pEGZ-term. The recombinant vector together with its helper virus vector were co-transfected into package cell 293T with Lipofectamine(TM); 2000.The supernatant of 293T was collected for infecting L929 cells (repeated three times), and cell clones stably expressing murine CXCR3 molecule were screened by zeocin(500 mg/L). We used FCM and RT-PCR to verify expression of CXCR3 from protein level and gene level, respectively. Studied migration ability of L929-mCXCR3 interacted with its ligand IP-10 by transwell system. RESULTS: We have constructed recombinant murine CXCR3 gene retroviral vector and obtained L929-mCXCR3 gene transfected cell line which can stably expressing murine CXCR3 molecule. Positive expression rate of membrane is 97.0%, and it can directly migrate induced by IP-10, the chemotatic index is 4.356%. CONCLUSION: Construction of L929-mCXCR3 cell line has laid a good foundation on research of biologic characteristics of CXCR3 signal path , establishment of tumor metastasis model and preparation of anti-murineCXCR3 monoclonal antibody.