| OBJECTIVE: The aim is to investigate the effect of PD-L1 ASODN on the liver metastasis of mouse colon carcinoma CT26 cell line.METHODS: The expression of PD-L1 in human colorectal carcinoma was measured by immunohistochemical S-P method.The number of CD4~+ and CD8~+ cells in human colorectal carcinoma was detected by immunofluorescecen. The correlation between PD-L1 and CD4~+, CD8~+ cells was analyzed. RT-PCR was used to detected the mRNA expression of PD-L1 in PD-L1 ASODN-transfected CT26 cells.Meanwhile, the liver metastasis of colorectal carcinoma model was established by intrasplenic inoculation of colon carcinoma CT26 cells in Balb/c mice. Two ways were used to treat the model with PD-L1 ASODN: liposomal transfection or intra-peritoneal administration. The mRNA expression of PD-L1 in tumor cells was determined by RT-PCR, and the number of liver metastastic tumor nudus was counted.RESULT: 1. PD-L1 positive staining rate (29/55, 52.7%)was higher in 55 cases colorectal carcinoma than that in control colorectal mucosa (1/10,10%) obviously (χ~2 test ,P<0.05). PD-L1 positive staining rate (22/33,66.7%)was higher in colorectal carcinoma with metastasis than that in colorectal carcinoma without metastasis (7/22,31.8%) (χ~2 test ,P<0.05).The PD-L1 expression was no related with the age, sex, pathologic grade, and the depth of infiltration(χ~2 test ,P>0.05). But the numbers of CD4~+ and CD8~+ T cell in PD-L1(+) cases was 12.241±3.181 and 25.931±4.390,separately. The numbers of CD4+ and CD8+ cell in PD-L1(-) cases was 29.077±4.524 and 68.539±5.405, separately. Spearman correlation analysis showed that the expression of PD-L1 was negative correlation with the numbers of CD4~+,CD8~+ lymphocytes infiltration (P<0.01).2. PD-L1 ASODN or RODN were transfeted into CT26 cell. The RT-PCR result showed that the ratio of PD-L1/β-actin in the two group were 0.041±0.002 and 0.524±0.022, the inhibition rate was 93% and 14.3%. separately.But the ratio of PD-L1/β-actin of control group (untreated group) was 0.612±0.015. Compared with RODN transfected group and control group, the PD-L1 expression was lower in ASODN transfected group(one way analysis of variance P<0.05).And inoculating the CT26 cell which transfected by PD-L1 ASODN or RODN into the Balb/c mice spleen. Then taking out of the primary tumors on spleen 14 days later.The PD-L1 expression of control group,RODN transfected group,PD-L1 ASODN transfected group were 0.615±0.010,0.505±0.005,0.14±0.007, the inhibition rate were 0%,17.9%,77%,separately. Compared with RODN transfected group and control group, the expression of PD-L1 was lower in PD-L1 ASODN transfected group (one way analysis of variance P<0.05).In the mice model of liver metastasis of colonl carcinoma CT26 cells, the numbers of metastatic carcinoma nodules on the liver surface of PD-L1 ASODN transfected group,RODN transfected group,control group were 6.833±4.021,41.500±8.916,46.000±11.541,separately. Compared with RODN transfected group and control group, the numbers of metastatic carcinoma nodules on the liver surface were less in PD-L1 ASODN transfected group(one way analysis of variance P<0.05).Besides,inoculating the CT26 cell in the BalB/c mice spleen and injecting PD-L1 ASODN or RODN into the abdominal cavity every 24h.14 days later ,the result shows that the ratio of PD-L1/β-actin of ASODN injected group was 0.197±0.008,but the RODN injected group was 0.528±0.008.And the inhibition ratio were 68%,14.1%, separately. There were significant difference when the rate of ASODN injected group was compared with that in control group or RODN group (one way analysis of variance,P<0.05).The numbers of metastatic carcinoma nodules on the liver surface of PD-L1 ASODN injected group,RODN injected group and control group were 7.500±1.871,41.833±7.083,46.000±11.541,separately . Compared with RODN injected group or control group, the numbers of metastatic carcinoma nodules on the liver surface were less than that in PD-L1 ASODN injected group (one way analysis of variance, P<0.05). But there was no significant difference between the two treatment ways (t test, P>0.05).CONCLUSION: The results suggest:1. The PD-L1 expression of human colorectal carcinoma was higher than that in control colorectal mucosa, and it related with metastasis ; 2. The PD-L1 maybe inhibit the CD4~+,CD8~+ lymphocytes gathering into the tumor in colorectal carcinoma. 3. PD-L1 ASODN can inhibit the expression of PD-L1. PD-L1 ASODN may inhibit the liver metastasis of colon carcinoma CT26 cells.The molecular mechanism need to be further study.
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