In Vitro Effects And Mechanisms Of Human DC Induced By SCD40l On Cervical Cancer Cell

Objective： To study the effects for soluble sCD40ligand on DCdifferentiation, maturation and immune activity of cervical cancer patients,and explore its mechanisms to provide a scientific basis for treating cervicalcancer in clinical.Methods:1The mononuclear cells (MNC) of cervical cancer patients wereseparated by density gradient centrifugation, cultured in RPMI1640mediumcontaining10%FBS for2h, and collected the adherent cells. The adherentcells were divided into three groups, concluding control group, TNF-α group,and sCD40L group. First, DCs were cultured by the cytokine GM-CSF(20ng/ml) and IL-4(20ng/ml), changed half of culture medium every one day,and cultured continuously for5days. In addition, TNF-α was added in TNF-αgroup, and sCD40L was used in sCD40L group respectively. Cells of allgroups were cultured for8d, and were collected for subsequent experiments.2DCs surface molecules, CD80, CD86, CD83, CD1a, MHCI and MHCII,were detected by FCM.3The expression of IFN-γ, IL-12, IL-10and TGF-β1on DCs wereanalyzed by RT-PCR, and levels of them detected in the culture system byELISA technology.4The effect of sCD40L on signal transduction pathway proteinsMAPKp38, P-STAT1and P-STAT3was detected by Western blot andRT-PCR respectively.5The effects of DCs on proliferation of allogeneic T cell were detected indifferent groups by MTT. The levels of IFN-γ and IL-4were analyzed byELISA technology in culture systems of different groups.6The killing activities of cytotoxicity T lymphocytes induced by DCs on Hela cells were detected by MTT method.Results:1The DCs of different groups have changed in morphology. In addition,the quantity of DCs in sCD40L and TNF-α groups increased obviously, andmorphology of cells was very typical.2Results of FCM showed that the expressions of CD80, CD86, MHCI,MHCII and CD1a of DCs surface molecule in sCD40L group were higher thancontrol group markedly (P<0.05), but compared to TNF-α group, there was nodisparation (P>0.05). However, the expressions of CD83in sCD40L groupand TNF-α group were higher than control group markedly (P<0.01), and theexpressions of CD83in TNF-α group was lower than in sCD40L group.3Results of RT-PCR revealed that mRNA levels of IFN-γ and IL-12ofDCs in sCD40L group were higher than control group (P<0.01). While mRNAlevels of IL-10and TGF-β1of DCs in sCD40L group were lower markedly(P<0.01).4Results of ELISA showed that compared to TNF-α group and controlgroup, the levels of IFN-γ and IL-12were higher in culture supernatant of insCD40L group (P<0.01). However, the levels of IL-10and TGF-β1werelower than control group (P<0.01), and the disparation was not meaningfulbetween TNF-α group and sCD40L group (P>0.05).5Results of RT-PCR and Western blot revealed that levels of MAPKp38、P-STAT1and P-STAT3of DCs in sCD40L group and TNF-α group werehigher than the control group markedly, and compared to TNF-α group, theabove three cytokines were higher in sCD40L group, which showed thatsCD40L played a role in differentiation and maturation of DCs throughactivating MAPKp38, P-STAT1, and P-STAT3signal pathway.6Results of MTT showed that T cells proliferation ability stimulated byDCs in sCD40L group was higher than control group obviously (P<0.01), butthe disparation was not meaningful between TNF-α group and sCD40Lgroup at ratio of1:80. However, T cells proliferation ability in sCD40L groupwas higher than TNF-α group (P<0.05) and control group (P<0.01) markedly at ratios of1:10or1:40.7Results of ELISA revealed that the levels of IFN-γ in sCD40L groupand TNF-α group were higher than control group (P<0.01), and compared tosCD40L group, the levels of IFN-γ in TNF-α group were more lower（P<0.05）. The levels of IL-4in sCD40L group and TNF-α group werehigher than control group（P<0.01）. There was no obvious disparationbetween sCD40L and TNF-α group（P>0.05）.8Results of MTT revealed that compared to control group, the killingactivities of cytotoxicity T lymphocytes induced by DCs on Hela cells werehigher at ratios of10:1or20:1(P<0.01). However the killing activities ofcytotoxicity T lymphocytes on Hela cells in TNF-α group and control groupwere lower than sCD40L group markedlyat ratios of50:1(P<0.05or P<0.01).Conclusion:1sCD40L can promote DC maturation and increase the expressin ofCD80, CD86, MHCI and MHCII, CD1a and CD83of DCs surface molecules.2Simulated by CD40L, the levels of IFN-γ and IL-12secreted by DCswere higher significantly and the levels of IL-10and TGF-β1much lowerobviously. The mechanism was associated with MAPKp38, P-STAT1,P-STAT3signal transduction pathway possibly.3sCD40L can significantly improve the proliferation of allogeneic Tlymphocyte induced by DCs, promote the secretion of IFN-γ by T cells andinhibit the secretion of IL-4.4DC stimulated by sCD40L can significantly promote lethal effect ofCTL on cervical cancer Hela.