ABSTRACT
Objective To construct lentiviral vectors containing peptide P1-GFP fusion genes.Umbilical cord derived mesenchymal stem cells were infected with lentivirus carrying peptide P1 and GFP fusion genes.To inject the targeted umbilical cord derived mesenchymal stem cells into mice and to detect GFP expression in the spleen.Methods Umbilical cord derived mesenchymal stem cells were cultured with adhered tissues of umbilical cord smaller than 1 mm3 . Lentiviral vector containing P1-GFP fusion genes with engineering technology was constructed and infected the umbilical cord derive mesenchymal stem cells.Targeted umbilical cord derived mesenchymal stem cells were intravenously injected in the mouse tail vein and after 18 hours GFP expression was detected with immunohistochamical staining of the spleen tissues.Results Harvested umbilical cord derived mesenchymal stem cells grew well in culture medium. Green fluorescence on umbilical cord derived mesenchymal stem cells were observed under fluorescence microscope at 18 hours after infected with lentivirus.Green fluorescence intensity of umbilical cord derived mesenchymal stem cells was increasing over time and reached a peak at 72 hours.Umbilical cord derived mesenchymal stem cells highly expressed CD105 (90.0%)/CD44 (98%) and CD73 (85.0%)/CD90 (98.5%) molecules.GFP expression was detected in the spleen after intravenous injection of targeted umbilical cord derived mesenchymal stem cells in the mice 18 hours later.GFP expressing cells intimately contacted with lymphocytes.Conclusions Targeted umbilical cord derived mesenchymal stem cells contain P1-GFP fusion genes are constructed.Targeted umbilical cord derived mesenchymal stem cells can be targeted to mouse spleen and intimately contact with lymphocytes after intravenous injection.Our results lay the groundwork for further studies.
ABSTRACT
<p><b>OBJECTIVE</b>To study the immune responses of lymphocytes after activated by dendritic cells (DCs) loaded with cytotoxicity T lymphocyte (CTL) epitope based peptide of human alpha-fetoprotein (hAFP, 218-226 LLNQHACAV).</p><p><b>METHODS</b>Get high purity DCs by cultured plastic-adherent monocytes isolated from healthy donor of HLA-A2(+) peripheral blood with granulocyte-monocyte colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) for 7 days. Stimulate self-lymphocytes with DCs that loaded with CTL epitope based peptide of hAFP under the culture medium contains interleukin-2 (IL-2) for 7 days. Analyse IL-12 and TNF in culture medium and also the specific lysis activity of lymphocytes against four strains of primary hepatocellular carcinoma cells.</p><p><b>RESULTS</b>After stimulated by DC loaded with CTL epitope based peptide derived from hAFP, lymphocytes appeared a good characteristics and the culture medium of activated lymphocytes contained a high level Th1 type cytokines of IL-12 and TNF. Activated lymphocytes not only specifically lysed HLA-A2(+) HepG2 line but also had the cytotoxicity against other three primary hepatocellular carcinoma cell lines and T2 target cell loaded with peptide of hAFP.</p><p><b>CONCLUSIONS</b>The results of this research supply the basic materials for the DC based vaccine with HLA-A2 restricted peptide epitope derived from hAFP against AFP positive primary hepatocellular carcinoma.</p>
Subject(s)
Adult , Animals , Humans , Mice , Dendritic Cells , Allergy and Immunology , Epitopes , HLA-A2 Antigen , Allergy and Immunology , K562 Cells , Peptides , Allergy and Immunology , T-Lymphocytes, Cytotoxic , Allergy and Immunology , Tumor Cells, Cultured , alpha-Fetoproteins , Allergy and ImmunologyABSTRACT
Objective:Got high purity dendritic cells from PBMC recovered from one time used no leukocytes transfusion apparatus.Methods:Rinsed PBMC with natural salt water from one time used no leukocytes transfusion apparatus and induced PBMC with GM-CSF and IL-4.Results:Got 5.1?10 7 PBMC from every one time used no leukocytes transfusion apparatus and 4.7?10 6 DCs with a purity of 96.9% were obtained.These DCs express CD86(96.1%)?CD40(88.9%)?CD83(91.6%)?HLA-DR(92.9%)?HLA-ABC(99%)?CD54(97.5%) highly and a few number of these DCs could stimulate proliferation of allogeneic mixed leukocyte reactions strongly.Conclusion:The results of this research supplied basic data for further research of DC and its clinical application.