Study on the effect of Toll like receptor signaling pathway on the regulation of dendritic cell maturation

Objective To explore the effect of TLR signaling pathway in the process of DC maturation. Methods Peripheral blood mononuclear cells( PBMC) were isolated using density gradient centrifugation. Immature dendritic cells( imDC) were induced by GM-CSF and IL-4 in vitro and were transfected with the total RNA extracted from A549 cells into imDC using electroporation. And then the imDC with A549-RNA transfacted were stimulated with traditional cocktail of cytokines( IL-1β,IL-6,TNF-α,PGE2) and modified cocktail of cytokines( IL-1β,IL-6,TNF-α,PGE2,polyⅠ∶ C,CpG ODN),respectively. The experiments were divided into four groups,control group: GM-CSF,IL-4; traditional group: traditional cocktail of cytokines; modified group 1: IL-1β,IL-6,TNF-α,polyⅠ∶ C,CpG ODN; modified group 2: IL-1β,IL-6,TNF-α,PGE2、polyⅠ∶C,CpG ODN. RNA transfection efficiency was detected by RT-PCR,surface markers of the DC and DC sensitized T cell were detected by flow cytometry,T cell proliferation was judged by MTT,the secretion of IL-12 were detected by ELISA. Results1) RT-PCR showed that the A549-RNA were transfected into the DC successfully by the method of electroporation,and these DC could express three tumor antigens,including tissue polypeptide specific antigen( TPS),carcino-embryonic antigen( CEA) and cytokeratin 19 fragment( Cyfra21-1). 2) Flow showed that CD83 expression rates in the above groups were( 15.17 ±1.61) %,( 64.33 ±7.58) %,( 60.26 ±4.18) % and( 82.89 ±3.06) %,CD1a expression rates were( 25.74 ±6. 06) %,( 69. 37 ± 8. 16) %,( 61. 66 ± 5. 82) % and( 86. 07 ± 5. 63) %,the proportion of CD8+T cells were( 36. 43 ± 3.15) %,( 61. 91 ± 2. 33) %,( 54. 81 ± 2. 55) % and( 66. 78 ± 2. 74) %,all these differences were statistically significant( P 0. 05). 3) The secretion of IL-12 in the above groups were( 55. 86 ± 14. 07) pg /mL,( 1 243. 49 ± 57. 57) pg /mL,( 744± 49. 51) pg /ml and( 2 385 ± 128. 98) pg /mL( P 0. 05). 4)Stimulation index in the above four groups and positive control group were 11. 85 ± 2. 61,54. 14 ± 3. 81,47. 57 ± 3. 14,64. 43 ±5. 88 and 135. 29 ± 5. 06( P 0. 05). Conclusion TLR ligand polyⅠ∶ C and CpG ODN can optimize the maturation of DC.