| Objective:To investigate the effect of tumor cells on transcription factor foxp3 and surface markers on CD4+Foxp3+Treg, analysis the role and evaluate possible mechanisms of Treg in immune evasion employed by cancer and its clinical significance.Methods:1. The percentage of CD4+Foxp3+Treg in CD4+T lymphocytes of peripheral blood in cancer patients and healthy donors were assayed by flow cytometry method to observe relative quantity of Treg, its surface markers GITR,CTLA-4,CCR4 and ICOS were also asssyed to observe whether their expression have changed in peripheral blood of cancer patients, and analysis the the association between them.2. Hepatocelluar carcinoma cancers patients were choosed from cancer patients as the objects of study, the association between relative quantity of Treg and AFP,ALT,AST in blood plasma of HCC patients were analysised, the association between surface markers GITR,CTLA-4 and AFP,ALT,AST were also analysised.3. PBMC were isolated by Ficoll-Hypaque density gradient centrifugation, PBMC were co-cultured with BEL7402, HepG2 and L02 cells in transwell chambers,and the total RNA was extracted from PBMC, Real-Time PCR was applied for the effect of Hepatoma cells supernatant on foxp3 mRNA transcription in PBMC of human peripheral blood.4. The secretion levels of TGF-βin cell supernatants of Hepatocellular carcinoma cell BEL7402,HepG2 and normol liver cell L02 were detected by ELISA method. PBMC were co-cultured with BEL7402 and L02 cells in transwell chambers,and Anti-human TGF-BAb was added simultaneously, Real-Time PCR was applied for the effect of Hepatoma cells supernatant on foxp3 mRNA transcription in PBMC of human peripheral blood after blocking TGF-β.Results:1. The percentage of CD4+Foxp3+Treg in CD4+T lymphocytes of peripheral blood in cancer patients was 20.25±14.22%, which was significantly higher than in normal controls, the latter was 6.96±1.38%(P<0.01).The levers of surface markers GITR (45.66±26.50%) and CTLA-4(41.66±22.90%) were increased in peripheral blood of cancer patients compared with normal controls(19.67±16.52 % and 19.24±11.94%, P<0.01), although the levers of CCR4 and ICOS were slightly higher than normal controls, there was no significantly defferences between them(P=0.177and P=0.058). There was also no association between the percentage of Treg and the levers of GITR,CTLA-4, but there was strong relevance between GITR and CTLA-4, and correlation coefficient was 0.479(P=0.007).2. There was strong relevance t between relative increasement of Treg in peripheral blood of HCC patients and AFP, the correlation coefficient was 0.610,(P=0.012),but there was no association between Treg and ALT,AST, and there were no correlation between GITR,CTLA-4 and AFP,ALT,AST.3. Foxp3 mRNA transcription in PBMC was increased obviously after PBMC were co-cultured with BEL7402, HepG2 cells in transwell chambers for 48hrs compared with normal control L02 cell(P<0.01).4. Hepatocellular carcinoma cell BEL7402,HepG2 can secret higher levels of TGF-βthan normal control L02 cell(P<0.05). PBMC were co-cultured with BEL7402, after blocking TGF-β, Foxp3 mRNA transcription in PBMC were decreased significantly compared with that not blocked.Conclusion:Tumor cells can promote the expression of transcription factor Foxp3 through secreting high levels of TGF-βand increase the frequency of Treg in peripheral blood of cancer patients, which resulting in the descent of immunity function in cancer patients, this may be one possible immune evasion mechanisms employed by cancer. At the same time, the levers of surface markers GITR and CTLA-4 were increased, may be they paly an important role in antitumor immunity response that Treg mediated. |
PDF Full Text Request