Expression And Correlation Of TREM-1 And Tumor-associated Macrophages In Patients With Primary Liver Cancer

Background and objective: Hepatocellular carcinoma is a kind of typical inflammation associated tumors.Studies have found that the triggering receptors expressed on myeloid cells(TREM)involved in immune regulation of inflammation,which playing a role in the pathogenesis of inflammation associated cancers.As the main immune cells in the tumor microenvironment,tumor associated macrophages(TAMs)play an important role in the invasion?metastasis and formation of blood vessels of tumor.The purpose of this study is to detect the expression of TREM/TAMs and the expression of TREM-1 on TAMs in HCC and the level of inflammatory cytokines such as sTREM-1?sCD163 and TNF-? in peripheral blood of patients with hepatocellular carcinoma.Besides,we established the M2 macrophage model in vitro,which laid the experimental foundation for further confirming the effect of TREM-1 on the differentiation phenotype of macrophages.Methods:20 primary liver cancer patients who were diagnosed in our hospital for surgery without any other treatment were taken as the research objects.The expression of sTREM-1,sCD163,TNF-?,CRP and IL-6 in the peripheral blood were determined by ELISA.The level of TREM-1 / TREM-2 mRNA was detected by qRT-PCR.Immunohistochemical staining was used to detect the expression of TREM-1 /TREM-2.Immunohistochemical double staining technique was used to detect the levels of M1 / M2 macrophages and the expression of TREM-1 on the surface of M2 macrophages.We isolated and cultured human peripheral blood monocyte cells in vitro,then determined the cell purity by flow cytometry;two methods,M-CSF,IL-4and GM-CSF were used to induced M2 macrophages and identify the phenotype of M2 macrophages by flow cytometry.The mouse bone marrow mesenchymal stem cells were also cultured in vitro,and were induced into macrophages with M-CSF,then the flow cytometry was used to determine the cell phenotype.Results:The level of inflammatory factors in peripheral blood including sTREM-1,sCD163 in patients with hepatocellular carcinoma were significantly increased(3.722 ± 0.02419 vs 2.606 ± 0.04036 ng/ml?2.093 ± 0.2835 vs 0.9379 ± 0.1326?g/ml,P<0.01),while TNF-?,CRP and IL-6 level had no significant difference between patients with hepatocellular carcinoma and patients with liver cirrhosis(P>0.05).TREM-1 mRNA levels in hepatocellular carcinoma tissues were significantly higher than that in paracancer tissues(0.1278 ± 0.01994,P<0.01),while the level of TREM-2 mRNA showed no significant difference between two groups(P >0.05).Immunohistochemical staining showed that the positive expression rate of TREM-1(11.23 ± 0.8840 %)in HCC tissues was significantly higher than that in paracancer tissues(2.253 ± 0.8662 %,P <0.01).The positive rate of M2 cells in HCC tissues(3.500 ± 0.5000 / HP)was significantly higher than that in paracancer tissues(0.5100 ± 0.1813 / HP,P<0.01),while M1 cells were not expressed.Immunohistochemical double staining showed that TREM-1(16.51 ± 0.9327 % vs6.644 ± 1.221 %,P <0.01)was highly expressed on the surface of M2 macrophages in hepatocellular carcinoma tissues compared to paracancer tissues.The positive rate of CD14 in monocytes that isolated and cultured was 83.3%.Compared with untreated monocytes,the positive rate of CD206 in M-CSF and IL-4 induced macrophages was significantly higher(63.57 ± 0.1202 % vs 0.7000 ± 0.05774 %,P <0.01),the positive rate of CD206 in macrophages induced by GM-CSF was significantly increased(92.87 ± 0.2906 % vs 0.7000 ± 0.05774 %,P <0.01).Compared with pre-induced bone marrow mesenchymal stem cells,F4/80 and CD11 b on M-CSF induced bone marrow mesenchymal stem cells were significantly increased(70.07 ± 0.1764 % vs1.533±0.1202 %,P <0.01).Conclusion:1)The phenotype of TAM in hepatocellular carcinoma was predominantly M2,TREM was expressed asTREM-1,and TREM-1 was expressed on the surface of M2 macrophages in HCC.2)The M2 macrophage model was successfully established in vitro.