| ObjectivesBy studying the expression of PD-1 and the function of CD4~+and CD8~+T cells induced by the exocytosis of hepatocellular carcinoma-derived exosomes,the relationship between the exocytic cells derived from hepatoma cells and the immune function of liver cancer was explored,which provided clinical immunotherapy for liver cancer.Theoretical basis.MethodsCollected in patients with hepatocellular carcinoma(North China university of science and technology affiliated hospital 2016.12 to 2018.05 in 52 patients with liver cancer has been diagnosed,28 cases male,female 24 cases,mean age 61.22±7.29 years)and healthy controls(North China university of science and technology affiliated hospital2016.12 2018.05 health check-up of 60 cases,32 cases of male,female 28 cases,the average age was 60.68±7.38 years)3mL of peripheral blood,blood taken on an empty stomach,through flow cytometry detection analysis of peripheral blood in patients with liver cancer and healthy controls the number of CD4~+,CD8~+T cells in difference,Meanwhile,PD-1 expression of CD4~+and CD8~+T cells in peripheral blood of liver cancer patients and healthy control group were analyzed.The mRNA expression of CD4~+and CD8~+T cells in peripheral blood of liver cancer patients and healthy control group was detected by q-PCR.In vitro,Huh-7 hepatocellular carcinoma cells were cultured and the supernatant was extracted.Exosomes were collected by precipitation method,and morphology and concentration particle size distribution of exosomes were detected by transmission electron microscopy and Nanosight.30mL of peripheral blood of healthy donors was collected,CD4~+and CD8~+T cells were separated and cultured by flow cytometry,and exosomes were added for 5 days to extract proteins.The protein expression of PD-1 in CD4~+and CD8~+T cells was detected by western-blot.SPSS 22.0statistical software was used for analysis.Data variance was homogeneity,and t test was used for comparison between groups.P<0.05 indicated statistically significant difference.ResultsThere was no significant difference in gender and age between liver cancer patients and healthy subjects.Flow detection of peripheral blood showed that the number of CD4~+T and CD8~+T cells(25.87±3.91%,14.81±3.34%)in peripheral blood of patients with liver cancer and the healthy group was not statistically significant(P>0.05)compared with the healthy group(28.28±3.96%,16.12±2.82%).Meanwhile,the number of CD4~+and CD8~+T cells expressing pd-1 in the peripheral blood of liver cancer patients was 8.32 1.81%and 9.38 1.72%,respectively,higher than that of the healthy group(2.57±0.99%and 2.75±1.08%,respectively),and the difference was statistically significant(P<0.05).CD4~+and CD8~+T cells in peripheral blood of liver cancer patients and healthy group were selected and RNA was extracted for q-PCR detection.MRNA expression levels of CD4~+and CD8~+T cells in peripheral blood of liver cancer patients were found to be 0.74 0.12 and 0.67 0.10,respectively,which were lower than those of healthy control group(1.11±0.14 and 1.18±0.12,respectively),and the differences were statistically significant(P<0.01).CD4~+PD-1~-and CD8~+PD-1~-T cells were isolated from peripheral blood of healthy subjects.The supernatant of Huh-7 hepatocellular carcinoma cells cultured in vitro was extracted,and exosomes were collected by precipitation method.A large number of concave disc-shaped exosomes were found by tem,and the average particle size of exosomes was about 150nm by nanosight detection.After the interaction of exosomes with the selected CD4~+PD-1~-and CD8~+PD-1~-T cells,the T cells were isolated to extract proteins.The protein expressions of pd-1 on the surface of the two kinds of cells detected by western-blot were 5.12±0.18 and 4.28±0.52,respectively,which were higher than those of the control group(1.04±0.24,1.02±0.32),and the difference was statistically significant(P<0.01).Conclusions1 There was no significant change in the number of CD4~+and CD8~+T cells in peripheral blood of patients with liver cancer compared with the healthy group.2The expression of CD4~+and CD8~+T cells in peripheral blood of liver cancer patients was significantly lower than that of the healthy group.3 The expression of CD4~+and CD8~+T cells pd-1 in peripheral blood of patients with liver cancer was significantly increased.4The expression of PD-1 in CD4~+and CD8~+T cells was significantly increased by exosomes derived from liver cancer cellsFigure 6;Table 6;Reference 182. |
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