Role Of TLR4/MyD88Signal Transduction Pathway In Fusion Vaccine By Peripheral Blood Dendritic Cells And Renal Cell Carcinoma786-0Cell Line

Objective： To investigate the change and function of toll-likereceptor4(TLR4)/Myeloid differentiation factor88(MyD88) signaltransduction pathway in fusion vaccine by peripheral blood dendritic cellsand renal cell carcinoma786-0cell line.Methods：Peripheral blood mononuclear cells were separated fromhealthy adults’peripheral blood by density gradient centrifugation,whichwas used to induce immature dendritic cells(imDCs),which were furtherinduced to fuse with renal cell carcinoma(RCC)786-0cells with aid ofpolyethylene glycol(PEG). Transmission electron microscope(TEM) wasemployed to observe the changes of imDCs before and after fusion;Semiqua-ntitative RT-PCR was used to detecte the mRNA chang of TOLL4and Myd88in pure imDCs, RCC786-0cells, and imDC-RCC786-0fusionvaccine in6,12,24,and48h after fusion. The transposition of nuclearfac-torkappa B(NF-κB) in imDCs before and after fusion was detected bylaser scanning confocal microscopy(LSCM); The chang of CD86andHLA-DR in imDCs before and after fusion were detected by flowcytometer; The abilities of the fusion vaccine to stimulate T lymphocytesproliferation and CTL cell mediated antitumor response were measured byMTT assay.Results： We obtained immature DC from peripheral bloodmononuclear cells,im DC and RCC786-O can be fused effectively by PEG.Transmission electron microscopy showed imDCs were transformedto mDC after fusion; The mRNA of TOLL4and Myd88was not expressedin RCC786-0cells,mildly expressed in imDCs,but was significantlystrongly expressed in fusion vaccine in6h after fusion(P＜0.05),then theirmRNA expression was decreased gradually till undetectable in48h afterfusin. LSCM displayed that NF-kB existed in the cytoplasm of pureimDC,and then translocated in the nucleus of fusion vaccine. Theexpressions of CD86and HLA-DR in fusion vaccine were signicantlyupregulated compared with the pure imDC control group(P＜0.05). Thefunctions of dendritic cell-tumor fusion vaccine to stimulate theproliferation of T lymphocytes were stronger than that ofimDCs,RCC786-0cells(P＜0.05),and CTLs stimulated by the fusionvaccine showed distinct activity in vitro to kill autologous tumor cells.Conclusion1.TLR4/Myd88-NF-κB signal transduction pathwayexhibits an very significance role in fusion vaccine to promote imDC toshift mDC, and the signal pathway prompts dendritic cells functionchanges;2.MDCs have more strong function than imDCs;3.MDCs can Continuous to provide Antigen information.