| AIM:Hepatocellular carcinoma(HCC)is the fifth most common cancer worldwide and so far no effective therapy has been achieved yet.Around HCC tumor there are various kinds of immunocytes,such as tumor-infiltrating lymphocytes(TIL)which are manifested as the strongest defense against malignant cells,and CD4~+ TIL has been recently recognized as more effective than CD8~+ TIL.However,a novel subgroup of CD4~+ T cells named regulatory T cell(Treg)has been found with immune suppressor function.Because Foxp3 is the specific marker for Treg,the suppressive CD4~+ T cell has been termed as Foxp3~+ Treg. But the mechanism of Foxp3~+ Treg is far to being understood.The presence of cyclooxygenase-2(Cox-2)is also closely associated with tumor growth,and it can suppress effecter lymphocytes in tumor-microenvironment.Some scholars have revealed that Foxp3~+ Treg might act to suppress immunity through Cox-2.So far however there have no reports on the research of the distribution of Cox-2,CD4~+ T cell and Foxp3~+ Treg in the tumor tissue,adjacent non-tumor tissue(ANT) and distant tissue in HCC,or on the correlation among Cox-2,CD4 T~+ cells and Foxp3~+ Treg in HCC.Combining with TGF-βllevel and clinical factors,in this study we investigated the distribution of Cox-2,Foxp3~+ Treg and CD4~+ T cells in the tumor tissue,adjacent non-tumor(ANT)tissue and distant tissue within the HCC tumor-microenvironment.At the same time,by using correlation analysis we analyzed the correlations of Foxp3~+ Treg,Cox-2 to CD4~+ T cells so as to find out the mechanism of Treg in the HCC tumor-microenvironment.METHODS:1.The test group consists of the tumor tissue,its matched ANT tissue(away from the solid tumor＜1cm)and matched distant tissue (away from the solid tumor＞5 cm)obtained from 40 HCC patients who all came from Hunan province,China,and all infected with hepatitis B virus.Also 5ml of venous blood was collected from each HCC patient.The control group consists of the normal liver tissue of 10 patients with hemangiomas and 5ml of normal adult venous blood.2.Methods:1)Cox-2 expression as well as the number of both CD4~+T cells and Foxp3~+ Treg is detected with immunohistochemistry.The relative quantitative SYBR Green RT-PCR is used for detecting the level of Foxp3 mRNA,while ELISA for the plasma TGF-β1 level. 2)Data on Cox-2,CD4~+ T cell,Foxp3~+ Treg and plasma TGF-β1 level are analyzed with patients' clinic figures,meanwhile the correlations among these four factors are analyzed by SPSS.RESULTS:1.In 40 HCC patients,the density of Cox-2 expression in order of decrease were ANT tissue,distant tissue,tumor tissue and normal tissue(P＜0.0001).Well-differentiated HCC expressed more Cox-2 than those in moderately and poorly-differentiated HCC(P=0.03)2.The number of CD4~+ T cells in order of decrease was ANT tissue, tumor tissue,distant tissue and normal tissue(P＜0.0001),and CD4~+ T cells was significantly higher in ANT than in other tissues(P＜0.0001).In the group of patients with Cox-2-expressing tumor,the number of CD4~+ T cells both in tumor tissue and in ANT tissue were lower than that in the other group with non Cox-2-expressing tumor(P＜0.0001).3.The number of Foxp3~+ Treg in order of decrease was ANT tissue, tumor tissue,distant tissue and normal tissue(P＜0.0001),and Foxp3~+ Treg was significantly higher in ANT than in other tissues(P＜0.0001).In the group of patients with Cox-2-expressing tumor,the number of Foxp3~+ Treg both in tumor tissues and in ANT tissues were higher than that in the other group with non Cox-2-expressing tumor (P＜0.0001).In ANT tissue of the 20 HCC,the Foxp3 mRNA was parallel to the number of Foxp3+ Treg detected with immunohistochemistry(P＜0.0001).4.In tumor and ANT tissues,statistically CD4~+ T cells showed a significantly negative correlation with Foxp3~+ Treg or Cox-2(P＜0.0001,P=0.003;P=0.002,P＜0.0001);Foxp3+ Treg was positively correlated with Cox-2(P＜0.0001,P=0.001).5.In HCC tumor-microenvironment,the more Cox-2,Foxp3~+ Treg or the less the CD4~+ T cells,the more possibility of venous metastasis existed;the more Foxp3~+ Treg or the less CD4~+ T cells,the higher serum AFP level appeared.Cox-2 expression showed no correlation to AFP level.6.The higher plasma TGF-β1 level,the more Cox-2 and Foxp3~+ Treg or the less CD4~+ T cells in HCC tumor-microenvironment was appeared (P=0.04,P=0.001).TGF-β1 level positively correlated to AFP level.CONCLUSIONS:1.The significantly more CD4~+ T cells are distributed in tumor tissue and ANT tissue than that in distant tissue and normal tissue.2.In HCC patients both Foxp3~+ Treg and Cox-2 are higher in tumor tissue and ANT tissue than that in normal control and they are negatively correlated to CD4~+ T cells,suggesting Foxp3~+ Treg and Cox-2 might act in a synergetic way to suppress CD4~+T cell.3.Foxp3~+ Treg and Cox-2 are positively related to the TGF-β1 level and AFP level,which might be a monitoring factor of prognosis or a therapeutic target in future clinical practice.
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