Construction Of Lewis Cell Line Carrying MGITRL Gene And Studies Of Its Biological Function

Objective:To construct eukaryotic expression vector containing mGITRL gene,named pIRES2-eGFP/mGITRL,and transfect it into Lewis cell. mGITRL and EGFP gene expressed stably in Lewis cell line.Approach effective of GITRL-GITR signal transduction pathyway in tumor therapy.Methods:(1)The mGITRL gene was amplified from intermediate vector PMD18-T/mGITRL by PCR and inserted into pIRES2-eGFP vector,and the sequence of DNA was analyzed.(2)pIRES2-eGFP/mGITRL vector was transfected into 4.0×10~6 Lewis cells by electroporation(0.22KV,960μf)and the positive clone was selected by G418 and subcloned.(3)CD4~+CD25~- T cells alone or mixed in equal amounts with CD4~+CD25~+ T cells,were stimulated for 3 days with anti-CD3 mAb and with mytomicin C-treated Lewis,eGFP- Lewis and GITRL-Lewis cells. Proliferation of CD4~+CD25~-T cells and suppressor function of CD4~+CD25~+T cells were detected respectively.(4)Injecting 200μl of PBS containing 2×10~6 Lewis,eGFP-Lewis and GITRL-Lewis cells subcutaneously into 6-week-old male C57BL/6 mice,living and tumor growth were monitored everyday.CD4~+CD25~+T cells proportion of tumor-bearing mice was analyzed,and proliferation of CD4~+CD25~-T cells and suppressor function of CD4~+CD25~+ T cells were also detected.Results:(1)Expression vector pIRES2-eGFP/mGITRL was constructed successfully,sharing 100%homology with the sequence of mRNA for mGITRL in GenBank(AY359852.1)(2)pIRES2-eGFP/mGITRL vector was transfected into Lewis cells by electroporation and the positive clone was selected by G418 and cloned,mGITRL gene and protein could be detected in the transfected Lewis cells.(3)Co-culture in vitro,the addition of mytomycin C-treated GITRL-Lewis cells greatly enhanced the proliferative capacity of CD4~+CD25~-T cells,and abrogated partly the suppressor of CD4~+CD25~+T cells.(4)Mice were injected GITRL-Lewis cells significant reduction of tumor size and prolonged survival of mice than mice injected Lewis and eGFP-Lewis cells.(5)Proportion of splenic CD4~+CD25~+T/CD4~+T in tumor-bearing mice is higher than normal mice.Proportion of CD4~+CD25~+T/CD4~+T in tumor-bearing mice TIL cells is higher than its splenocyte.Proportion of CD4~+CD25~+T/CD4~+T in GITRL-Lewis tumor-bearing mice TIL is losser than Lewis,eGFP-Lewis tumor-bearing mice.(6)Proliferation of CD4~+CD25~-T cells of tumor-bearing mice is losser than normal control mice,but proliferation of CD4~+CD25~-T cells of GITRL-Lewis tumor-bearing mice is higher than eGFP-Lewis tumor-bearing mice.CD4~+CD25~+ T cells of tumor-bearing mice can suppress proliferation of CD4~+CD25~-T cells of normal mice,but the suppressor of CD4~+CD25~+T cells of GITRL-Lewis tumor-bearing mice is losser than eGFP-Lewis tumor-bearing mice.Conclusions:(1)Expression vector pIRES2-eGFP/mGITRL has been constructed successfully.(2)mGITRL was expressed stably in Lewis cell.(3)Mytomicin C-treated GITRL-Lewis cells can enhance the proliferative capacity of CD4~+CD25~-T cells,and abrogate partly the suppressor of Treg cells.(4)Tumorigenesis capability of GITRL-Lewis cells is losser than wild type Lewis cells.