ABSTRACT
<p><b>OBJECTIVE</b>To study the cytotoxic activity against tumor cells and cytokines production of spleen cells induced in vitro by murine 4-1BBL gene transfected Hepa1-6.</p><p><b>METHODS</b>The eukaryotic expression vector pCDNA3.1(+)-m4-1BBL was transfected into murine hepatocellular carcinoma cell line Hepa1-6 by Liposomes. Then the transfected cells were selected in medium containing G418 (400 - 800 micro g/ml) and termed as Hepa1-6-m4-1BBL. The TCV-m4-1BBL was obtained by treating them with mitomycin (MMC). Cocultivation TCV with syngeneic murine spleen cells, then the lymphocytes were tested for cytotoxic activity against Hepa1-6-wt cells and the supernatants were harvested for detecting the cytokines (IL-2, TNF-alpha and GM-CSF).</p><p><b>RESULTS</b>Hepa1-6-m4-1BBL cells expressed 4-1BBL protein with highest cell surface level. The 4-1BBL mRNA could still be detected in the cells when cultured 48 h after treated with MMC (80 mg/L). Comparing with TCV-Hepa1-6, the tumor cell vaccine derived from Hepa1-6-m4-1BBL (TCV-m4-1BBL) could induce a more efficient cytotoxic activity of syngeneic murine lymphocyte against its parental tumor cell Hepa1-6 (P < 0.05), but not against non-parental tumor cell H22 and NIH3T3. Higher levels of IL-2, TNF-alpha and GM-CSF were released by the splencytes after stimulated by TCV-m4-1BBL.</p><p><b>CONCLUSIONS</b>These results suggest the expression of m4-1BBL by tumor cells is effective in inducing antitumor immune responses.</p>
Subject(s)
Animals , Female , Mice , 4-1BB Ligand , Cancer Vaccines , Allergy and Immunology , In Vitro Techniques , Liver Neoplasms, Experimental , Allergy and Immunology , Mice, Inbred C57BL , T-Lymphocytes, Cytotoxic , Allergy and Immunology , Transfection , Tumor Necrosis Factors , Genetics , PhysiologyABSTRACT
Using the isolated total RNA from osteosacoma cell line MG63, the cDNA encoding human OPG was amplified by RT-PCR. A recombinant adenoviral vector carrying cDNA of OPG was constructed and OPG expression in mouse myoblast C2C12 cells was confirmed by Western blot and ELISA. The secreted expression of OPG protein persisted more than 6 weeks in vitro, and the growth of C2C12 cells infected by recombinant adenoviral were in good state. Osteoclasts derived from mouse bone marrow cells infected with recombinant adenoviral made less number of TRAP positive cells and resorption pits formed on dentine slices.