ABSTRACT
<p><b>OBJECTIVE</b>To construct a retroviral-mediated vector of FLT3 ligand (FL) and express it in human bone marrow stromal cells.</p><p><b>METHODS</b>FL cDNA was inserted into the retroviral vector pLXIN by gene recombination technology. The recombinant plasmid pLFIN was transferred into retrovirus packaging cell line PA317 by lipofectamine, and the positive clones were selected by G418. The mRNA expression in human stromal cells and integration of genome DNA were assayed by reverse transcriptase-polymerase chain reaction (RT-PCR) and genomic DNA-PCR. The expression of FL protein and its biological activities in the culture were investigated by ELISA and mouse bone marrow CFU-GM assay.</p><p><b>RESULTS</b>The recombinant plasmid pLFIN was successfully constructed. In genome of these transfected target cells, Neo gene and FL gene were integrated, FL mRNA was transcripted and FL protein was expressed at 4.35 ng/ml/24 h. The specific activities of FL in the culture indicated that human bone marrow stromal cells transfected with FL could significantly express FL in vitro.</p><p><b>CONCLUSION</b>The retroviral-mediated FL gene was expressed in bone marrow stromal cells and the biological activities of FL were detectable in the supernatant of the transfected cells. These results provide a basis for studies on hematopoietic regulation by gene transfected bone marrow stromal cells.</p>
Subject(s)
Animals , Humans , Mice , 3T3 Cells , Bone Marrow Cells , Cell Biology , Metabolism , Cell Line , Colony-Forming Units Assay , Enzyme-Linked Immunosorbent Assay , Gene Expression , Genetic Vectors , Genetics , Membrane Proteins , Genetics , Metabolism , RNA, Messenger , Genetics , Metabolism , Retroviridae , Genetics , Reverse Transcriptase Polymerase Chain Reaction , Stromal Cells , Cell Biology , Metabolism , TransfectionABSTRACT
Objective: To explore antitumor responses induced by recombinant vaccinia viruses expressing a p53 antigenic peptide (rVV-p53M) and enhanced effect of recombinant vaccinia viruses expressing costimulatory molecule B7 (rVVB7). Methods: A 135 Cys to Tyr point mutation p53-transduced P815 mastocytoma (P815-mp53) was used as an experimental tumor and the p53 protein as the model of tumor associated antigen． rVV-p53M and rVV-B7 were used as vaccine to test their induction of CTL and antitumor immunity. Results: Immunization of BABL/c mice with rVV-p53M could elicit specific CTLs, which could specifically lyse P815-mp53 cells. Immunization of mice with rVV-p53M could survive a part of mice challenged with 1×106 P815-mp53. Treatment with rVV-p53M could significantly prolong the survival oftumor-bearing mice. Admixture at 1:1 ratio of rVV-p53 M and rVV-B7 could enhance antitumor responses induced by rVV-p53M. Conclusion: Immunization with recombinant vaceinia virus expressing antigenic peptide is a useful alternative to peptide-based vaccine. Costimulatory molecule B7 can enhance antigenic peptide to induce antitumor responses.
ABSTRACT
Objective: To explore antitumor responses induced by recombinant vaccinia viruses expressing a p53 antigenic peptide (rVV p53 M) and enhanced effect of recombinant vaccinia viruses expressing costimulatory molecule B7 (rVV B7). Methods: A 135 Cys to Tyr point mutation p53 transduced P815 mastocytoma (P815 mp53) was used as an experimental tumor and the p53 protein as the model of tumor associated antigen. rVV p53 M and rVV B7 were used as vaccine to test their induction of CTL and antitumor immunity. Results: Immunization of BABL/c mice with rVV p53 M could elicit specific CTLs, which could specifically lyse P815 mp53 cells. Immunization of mice with rVV p53 M could survive a part of mice challenged with 1?10 6 P815 mp53. Treatment with rVV p53 M could significantly prolong the survival of tumor bearing mice. Admixture at 1∶1 ratio of rVV p53 M and rVV B7 could enhance antitumor responses induced by rVV p53 M. Conclusion: Immunization with recombinant vaccinia virus expressing antigenic peptide is a useful alternative to peptide based vaccine. Costimulatory molecule B7 can enhance antigenic peptide to induce antitumor responses.