| OBJECTIVE:To evaluate the antitumor effect and immune mechanism of rAd-mIL-28B,the subcutaneous transplantation tumor model of H22 hepatoma cells in Balb/C strain mice was employed.METHODS:Human embryonic kidney cells(HEK293A cells)were cultured in conventional methods,and HEK293A cells were infected with rAd-mIL-28B and rAd-EGFP adenovirus stock solution to amplify the two viruses.The titers of rAd-mIL-28B and rAd-EGFP viruses were meassured according to cytopathic effect(CPE),H22 hepatoma cells were cultured in vitro and in mouse peritoneal cavity to establish a subcutaneous xenograft model of H22 hepatoma cells in Balb/C mice,the experiment was divided into untreated group(Untreated group),rAd-mIL28B group,rAd-EGFP group and cisplatin group(Cisplatin group).The size and body weight of transplanted tumor in mice were measured every day.After 10 days of drug treatment,the mice were sacrificed,the tumor tissues,spleens and livers were weighed,and the tumor growth inhibition rate(tumor inhibition rate),spleen index and liver index were calculated.The pathological changes of tumor tissues were observed by HE staining,the percentages of CD4~+T cells,CD8~+T cells and PD-1~+T cell subsets in spleens of mice were detected by flow cytometry,the levels of serum IFN-γand IL-10 in mice were detected by ELISA.RESULTS:1.The spleen index of the untreated group,rAd-mIL-28B group,rAd-EGFP group and cisplatin group were 79.60±13.61,128.30±8.55,96.33±28.21,57.58±19.99,respectively.The treatment of rAd-mIL-28B led to a significant increase of spleen index compared with rAd-EGFP(P<0.05).The liver index of the untreated group,rAd-mIL-28B group,rAd-EGFP group and cisplatin group were68.67±7.14,70.85±9.58,64.67±8.31 and 67.18±9.89 and there was no statistically significant difference in liver index among all groups(P>0.05).2.The tumor weights of the untreated group,rAd-mIL-28B group,rAd-EGFP group,and cisplatin group were 2.08 g±0.636 g,2.34 g±0.254 g,3.96 g±0.387 g,and 1.86 g±0.351 g,respectively.Compared with the EGFP group,the tumor inhibition rates of the rAd-mIL-28B group and the cisplatin group were 40.8%and52.9%,respectively.In general,rAd-mIL-28B significantly inhibited the growth of H22 hepatoma cell xenografts.3.HE staining showed that that the tumor cells in the untreated and rAd-EGFP groups were tightly packed with high cell density,and that no large areas of tumor cell necrosis were observed.Varying degrees of tumor cell necrosis were detected in the tumor tissues in the rAd-mIL-28B and cisplatin groups,with significant cellular atypia and low cell proliferation density.The percentages of CD4~+T cells in the untreated group,rAd-mIL-28B group,rAd-EGFP group and cisplatin group were:4.17%±0.85%,10.82%±1.44%,10.76%±4.54%,and 15.39%±3.94%respectively.The percentages of CD4~+T cells in rAd-mIL-28B group,rAd-EGFP group and cisplatin group were higher than that in the untreated group(P<0.05).The percentages of CD8~+T cells in the untreated group,rAd-mIL-28B group,rAd-EGFP group and cisplatin group were:1.83%±0.39%,2.47%±0.31%,2.59%±0.93%,and 4.68%±1.76%,respectively.The percentages of CD8~+T cells in rAd-mIL-28B group,rAd-EGFP group and cisplatin group were significantly higher than that in the untreated group(P<0.05).rAd-mIL-28B up-regulates and promotes the proliferation of T lymphocytes.4.The percentages of CD4~+PD-1~+T cells in the untreated group,rAd-mIL-28B group,rAd-EGFP group and cisplatin group accounted for 1.93%±0.19%,0.96%±0.11%,1.67%±0.36 and 1.67%±0.30%.The percentages of CD8~+PD-1~+T cells in the untreated group,rAd-mIL-28B group,rAd-EGFP group and cisplatin group were0.11%±0.01%,0.07%±0.01%,0.12%±0.03%and 0.14%±0.05%.In general rAd-mIL-28B reduced the percentage of CD4~+PD-1~+T cells and CD8~+PD-1~+T cells(P<0.05)and inhibited the expression of PD-1 molecules.5.The average concentrations of serum IFN-γin untreated group,rAd-mIL-28B group,rAd-EGFP group and cisplatin group were 22.70 pg/mL±8.74 pg/mL,252.49pg/mL±3.28 pg/mL,46.65 pg/ml±22.66 pg/mL and 118.98 pg/mL±108.20 pg/mL.The mean concentrations of serum IFN-γin the rAd-mIL-28B group and the cisplatin group were significantly higher than those in the untreated group and the rAd-EGFP group(P<0.05),suggesting that rAd-mIL-28B can promote the body’s secretion of IFN-γ.The average concentrations of serum IL-10 in the untreated group,rAd-mIL-28B group,rAd-EGFP group and cisplatin group were 13.40 pg/ml±3.27pg/mL,13.24 pg/mL±1.99 pg/mL,21.28 pg/mL±6.50 pg/mL and 9.18 pg/mL±3.43 pg/mL and there was no significant difference between the groups(P>0.05).CONCLUSIONS:rAd-mIL-28B can effectively inhibit the growth of subcutaneous transplantation tumor of H22 hepatoma cells,induce proliferation and activation of CD4~+T cells and CD8~+T lymphocytes and up-regulate IFN-γin serum.Furthermore,rAd-mIL-28B can reduce the percentages of CD4~+PD-1~+T cells and CD8~+PD-1~+T cells in mouse spleen.In general,the possible anti-tumor mechanisms of rAd-mIL-28B were inhibiting PD-1 molecule expression and promoting T cell activation. |
PDF Full Text Request