Studies On Induction Of Dentritic Cell By Adeno-Associated Virus In Vitro

Objective: The peripheral blood mononuclear cells were infected with three kinds of adeno-associated virus containing cytokine gene ( GM-CSF,IL4 and TNFα) in turn,while corresponding cytokines were secreting,thus DCs were inducing.Methods: By co-transfecting the helper plasmid,pHELPER ,and the vector, rAAV/cytokine containing the cytokine cDNA(GM-CSForIL4 or TNFα)into the packaging cell line 293 cell ,concentrated crude virus stocks were prepared from infected 293 cells by twice cycles of freeze-thawing..The titers were calculated by dot blot .The PBMC were separated from fresh blood drawn of healthy volunteers by routine Ficoll gradient method.The PBMC cells were infected with different AAV/cytokine virus stocks,and the different cytokine mRNA expression was analysised by RT-PCR amplification.The PBMC were separated drawn from healthy volunteers again.The PBMC were dealed with three methods,infected with three kinds of AAV/cytokine virus stocks(AAV/GM-CSF,AAV/IL4 and AAV/TNF)or cultured withexogenous cytokines(rhGM-CSF,rhIL4 and rhTNFα) or only cultured with 293 cells lysate respectively at the same time.These three groups cells are analyzed on morphology,cellular structure under optics and electronic microscope,and then phenotype of cells were detected by FACS analysis including molecules marker CD14,CD40,CD83,CD86;and was performed by MLR and MTT to observ the ability of DC stimulating lymphocytes proliferation.Results: (1) The titers of virus were determined to be about 1×107-8 encapsidated genomes per ml(eg/ml).(2)The different cytokine mRNA expression in PBMC was detected by RT-PCR amplification ,and it's products were visualized by ethidium bromide staining on an ultraviolet light transilluminator.(3)Typical DCs can be induced when PBMC were infected with AAV/cytokine in turn at day 10.(4)The detection of MLR shows that DCs can stimulate strong T lymphocytes proliferation.Conclusion: (1) rAAV can successfully transfer the foreign gene into the eukaryotic cell,it's direct genetic alteration should result in continuous,stable expression of foreign gene. (2)Typical DCs can be induced with gene trasfer. (3) It should provide the experimental foundation for searching for a more optimize method of induction DC.