| Objective: Dendritic cell-derived exosomes(DEX) is a kind of membrane structure of DC, which obtains main functional proteins from DC cells and can induce the active immune responses. In this experiment, we tried to collect exosome from DC infected with rh AAV/AFP, and explore the effect of DC-derived exosome(DEX)on activing T lymphocytes and killing hepatocarcinoma.methods: 1. Peripheral blood mononuclear cells of healthy donors were induced with IL-4, GM-CSF and LPS.The morphology of the cells was observed under microscope and the functional molecules expression of DC was identified by flow cytometry.2. Infection of rh AAV/AFP virus was observed under fluorescence microscope and the efficiency of infection was measured by flow cytometry.3. Exosome was collected and purified by ultrafiltration and sucrose density gradient centrifugation. The morphology of DEX was observed under the transmission electron microscope. The size of DEX particle was detected by Nano ZS90(Malvern), and the proteins in DEX were checked by using western bloting of its functional protein and liver cancer antigen expression.4. DC was loated with the recombinant adenovirus with AFP gene(rh AAV/AFP)and the exosome derived from rh AAV/AFP loated DC(DEXrh AAV/AFP) respectively.5. The proliferation effciency of T lymphocyte stimulated by(A) DC-rh AAV/AFP,(B) DEXrh AAV/AFP,(C) DC-DEXrh AAV/AFP,(D) DC-Control respectively was detected by CFSE staining method and observed under fluorescent microscope.6. The viability of naive T cells and the expression level of functional molecules and secretion of cytokines were assessed by FCM. 7-AAD /Annexin V double staining method was used to detect the killing effect of CTL on AFP positive Hep G2 cells and AFP negative SMMC7721 cells in each group.Results: 1. m DC showed typical dendritic cell morphology, and its surface molecule CD83, CD1 a, CD80, CD86 expression was significantly higher than that on im DC(P <0.05), but no difference in the expression of CD209(ICAM-3) and HLA-DR was found(P> 0.05).2. The infection efficiency of DC with rh AAV/AFP reached 86% by green fluorescence analysis.3. Under the transmission electron microscopy(TEM), DEXrh AAV/AFP extracted by ultrafiltration and density gradient ultracentrifugation showed 50 nm to 100 nm in diameter for round or oval corpuscle. DEX expressed exosome membrane molecular including CD63, CD9, DC function related molecules including ICAM-1,HLA- DR, CD86, HSP70, as well as liver cancer antigen AFP molecules.4. DC-rhAAV/AFP(A)?DEXrh AAV/AFP(B)?DC-DEXrh AAV/AFP(C) stimulated T lymphocyte proliferation was significantly higher than DC control(D)(P <0.05).5. CD45 RO, LFA-1a / CD244, Granzyme B, Perforin expression of the effector cells induced by DC-rh AAV/AFP(A), DEXrh AAV/AFP(B) and DC united DEX—DC-DEXrh AAV/AFP(C) were significantly higher than the DC control group(D)(P<0.05). CD28, OX40 HLA-DR expression of effector cells induced by DC-DEXrh AAV/AFP(C) were significantly higher than the control group(D)(P <0.05).CD45 RA expression in DEXrh AAV/AFP and DC-DEXrh AAV/AFP group was significantly lower than that in the control group(P<0.05). PD-1 expression of DC-DEXrh AAV / AFP was significantly higher than that in control group or DC-rh AAV/AFP(P <0.05). No significant difference of the expression of CD69,CD71, CD95, CTLA-4 was found among the four groups(P> 0.05). IFN-? level in DC-DEXrh AAV/ AFP group was significantly higher than the control group(P>0.05).Compared with other groups, IL-4 and IL-17 A levels of DEXrh AAV/AFP group were significantly lower(P <0.05).6. 7-AAD/Annexin-V double staining was used to determine the killing effect of effector cells induced by DC and DEX in vitro against hepatocellular carcinoma cell line Hep G2, SMMC-7721.Under the conditions of effector/target ratio of 25: 1,DC, DEX and DC-DEX induced CTL got obvious cytotoxic effect on hepatoma cell lines, significantly higher than the DC control group(P<0.05). The cytotoxicity of CTL induced by DC, DEX and DC-DEX to AFP positive Hep G2 were showed with(70.93±3.08)%,(71.16±4.74)%,(75.33±6.30)% respectively, and significantly higher than that to the AFP negative SMMC-7721 cell lines((49.70±2.55) %,(51.26±4.11) %,(53.13±2.47)(P<0.05)). Killing rate of CTL induced by DC-DEX on liver cancer cell slightly higher than the DC and DEX, and there was no significant difference between CTLs induced by DC and DEX(P >0.05).Conclusion:1. DC infected by rh AAV/AFP can sustain expression of AFP gene efficiently in DC-mediated immune activity and induced an effective hepatocellular carcinoma-specific CTL effect. Adeno-associated virus which carry tumor antigen gene could be regarded as an effective way of preparation of DC vaccine.2. Ultrafiltration combined sucrose density gradient ultracentrifugation is an effective and simple method of separation of DEX with high purity. m DEX expression of functionally related molecules, exosomes marker and tumor antigen molecules.3. As a cellular vaccine having an antigen-presenting role, DEX can stimulate the initial activation and proliferation of T lymphocytes and plays a specific role on anti-tumor immune response. DC loaded with DEX got a synergistic effect. |
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