Suppression Effect Of Co-inhibiting PD-1 And CTLA-4 Expression On H22 Hepatomas In Mice

Objective: The aim of this study is to transfect si RNA into tumor bearing mice by RNA interference technology,thereby down regulating the expression of PD-1 and CTLA-4 in mice,and further explore the suppression effect of co-inhibiting the PD-1 and CTLA-4 expression on H22 hepatoma by si RNA in mice.Methods: Murine H22 cell was cultured in vivo of ICR mouse and xenograft tumor model was established in another ICR mouse.The tumor-bearing mice were randomly divided into four groups: control,single PD-1 si RNA,single CTLA-4 si RNA and double PD-1+CTLA-4 si RNAs.The survival time and physiological condition of mice were observed after injecting si RNAs and placebo.The volume and weight of solid tumor was measured to assess the inhibition of the tumor.The protein levels of IFN-? and IL-10 in blood,PD-L1 in tumor and liver were detected by ELISA.The expressions of IFN-?,PDL1,IL-6 and Survivin in tumor tissue,m RNA expressions of IFN-?,PD-L1 in liver tissue,IFN-?,PD-1 and CTLA-4 in spleen tissue were detected by q PCR to analysis of m RNA expression of each gene on the m RNA level.The relative expression of Bax and Bcl-2 protein was detected by Western Blot.The ratio of gray value of Bax and Bcl-2 protein was used to analyze the effect of si RNA on the apoptosis of tumor cells.Results: The anti-tumor effect appeared in all inhibiting groups.The tumor growth suppression was stronger in the double inhibiting group.The survival rate of bearing tumor mice was 100% in double si RNAs,75% in PD-1,71% in CTLA-4 and 30% in control group.The experimental results showed that the relative expression of PD-L1 m RNA and protein in the three administration groups increased significantly,but decreased in the liver tissue(P<0.05).Compared with the control group,IFN-? in the blood of inhibitory group mice increased,and IL-10 was reduced(P<0.05).After using interfering RNA,the relative expression of PD-1 and CTLA-4 in spleen tissue was down-regulated(P<0.05).And the expression of IFN-? in tumor and spleen tissues was significantly increased,while the expression of IFN-? in liver tissue was significantly decreased(P<0.05).In tumor tissue,the relative expression of survivin and IL-6 m RNA was decreased(P<0.05).Western blot analysis of Bax protein and Bcl-2 protein gray value ratio was significantly increased(P<0.05).Conclusions: co-inhibiting expression of PD-1 and CTLA-4 can effectively suppress the growth of H22 hepatoma,promote the apoptosis of tumor cells in mice.Blocking PD-1 and CTLA-4 can improve the vitality of T cells,improve the immune environment and the immunity of body.