Effects Of Hepatocellular Carcinoma Cells' Apoptosis And The Related Mechanisms After Indoleamine 2,3-dioxygenase Gene Transfection.

Objects Hepatocellular carcinoma patients' immune systems cannot effectively remove or kill cancer cells, it is one of the important factors to cause that liver cancer are incurable.Indoleamine 2,3-dioxygenase(IDO) catalyzes the rate-limiting step of tryptophan degradation along the kynurenine pathway,and also can make the body to produce acquired immune tolerance. Many researches show that in multiple tumour tissues high expression of IDO has relations with the growing number of Treg cells,so IDO and Treg cells are expected to become potential biological treating targets. This study is to investigate the relationship of IDO high expression and the number of Treg cells,and the effects of hepatumor cells and T lymphocytes in the environment of hepatocellular carcinoma. These will provide a new theory basis and clinical evidence for liver cancer immunotherapy treatment.Methods By cell culture and gene transfection technology,T-lymphocytes freshly isolated from healthy people and hepatocellular carcinoma cells were cocultured. The experiments were divided into six groups: T-lymphocytes and HepG2 cells group, T-lymphocytes and pcDNA3.1-HepG2 cells group, T-lymphocytes and pcDNA3.1-IDO-HepG2 cells group ,and the three intervention groups which were added to IDO inhibitor 1-MT(1-methyl-tryptophan) on the basis of the above three groups. After two days of combine reaction, the apoptosisrate of HepG2 cell and the cytotoxicity of T-lymphocyte against HepG2 cell were examined by flow cytometer and MTT assay. After five days in mixed culture,the percentage of regulatory T cells(Treg) were analyzed by flow cytometer. Through establishment of the mouse model of human liver cancer cells, the percentage of Treg cells in peripheral blood of mouse was analyzed by flow cytometer.Results 1. After two days of combine reaction , the earlier apoptosis rate of HepG2 cell in IDO-HepG2 group was significantly lower than which in pcDNA3.1-HepG2 and HepG2 groups. They were respectively(1.65±0. 14) %, (7. 17±0. 24) %,(6.39±0. 33) % ( P < 0. 05) .However, with 1-MT groups, they were respectively (4.41±0. 70) %, (10.50±1.51) % ,(11.55±1.05) %,it was considered statistically significant compared with the control group without 1-MT.2. The cytotoxicity of T-lymphocyte against HepG2 cell in control groups were respectively ( 35.00±2.20) % , ( 74.51±1. 44) % , ( 75.30±1. 67) % ( P < 0. 05);and with 1-MT groups, that were respectively (71.33±2.10)%,(81.27±2.42)%,(80.91±1.95)%; there was a significant increase in the cytotoxicity of T-lymphocyte against HepG2 cell in cocultured condition with 1-MT groups.3.After five days of combine reaction, the percentage of Treg cell in IDO-HepG2 group was significantly higher than which in pcDNA3.1-HepG2 and HepG2 groups. They were respectively(10.53±1.05)%,(4.03±0.17)%(,3.34±0.18)% ( P < 0. 05);in adding 1-MT groups,that were respectively (6.37±0.15)%,(1.43±0.10)%,(1.57±0.13)%, it was considered statistically significant compared with the control group without 1-MT.4.In the mouse model of human liver cancer cells,the percentage of Treg cell in peripheral blood of IDO-HepG2 group was significantly higher than which in pcDNA3.1-HepG2 and HepG2 groups; they were respectively(15.33±1.18)%,(8.96±0.53)%,(8.14±0.57)%; it was considered statistically significant between the over- expresstion of IDO group and other groups(P< 0. 05).Conclusions 1,Though increasing the percentage of Treg cells in T-lymphocytes, IDO can suppress the apoptosis rate of hepatocellular carcinoma cells and the cytotoxicity of T-lymphocyte. 1-MT can reverse the role of IDO. 2,In vivo test, it can be confirmed that over-expression of IDO can increase the proportion of Treg cells in peripheral blood.