Effects of Hepatocellular Carcinoma Cells’ Apoptosis and the Related Mechanisms After Indoleamine 2,3-dioxygenase Gene Transfection.
|Course||Department of Gastroenterology,|
|Keywords||Indoleamine 2 3 - dioxygenase Hepatoma cells T lymphocytes Treg cells Apoptosis|
Purpose of the immune system of patients with liver cancer can not effectively remove or kill cancer cells, leading to liver cancer is difficult to cure, recurrence and metastasis is an important factor. Indoleamine 2,3 - dioxygenase (indoleamine 2,3-dioxygenase, IDO) allows the body to produce an acquired immunological tolerance of the enzymes, but also the only place outside the liver can catalyze the oxidative cleavage of tryptophan limit Speed ??enzymes. Studies have shown that in a number of tumor tissue with high expression of IDO regulatory T cells (Regulatory T cell, Treg) infiltration related increase in the number, the IDO and Treg cells is expected to become potential biological therapeutic targets. This study aimed to investigate the liver environment with high expression of IDO relationship between the number of Treg cells, as well as liver cells and T lymphocytes role for immune therapy of liver cancer biology and provide new theoretical basis and clinical basis. Extracted healthy human peripheral blood T cells using cell culture and gene transfection technique T cells and liver cancer cells mixed culture. Were divided into six groups: According to whether to join the D-1-MT is not divided into intervention group and intervention groups, each based on different cultured cells and T cells and HepG2 cells were divided into groups, T cells and pcDNA3.1-HepG2 cell group , T cells and pcDNA3.1-IDO-HepG2 cell group. 2 days in mixed culture by flow cytometry, MTT assay in each group HepG2 cell apoptosis in HepG2 cells and T cells against cytotoxic activity. 5 days in a mixed culture of Treg cells by flow cytometry proportion. And the establishment of a mouse model of human liver cancer cells using flow cytometry in peripheral blood of tumor-bearing mice proportion of Treg cells. Results 1, FACS analysis mixed reaction system HepG2 cells apoptosis rate, not in the intervention group with high expression of IDO pcDNA3.1-IDO-HepG2 cell apoptosis was significantly lower than pcDNA3.1-HepG2 cells and normal HepG2 cells, respectively (1.65 ± 0.14)%, (7.17 ± 0.24)%, (6.93 ± 0.33)%, the former with the latter two statistically significant difference (P lt; 0.05); while the intervention group withered HepG2 cells Death rates were higher than before the intervention, namely (4.41 ± 0.70)%, (10.50 ± 1.51)%, (11.55 ± 1.05)%, before and after intervention was statistically significant (P lt; 0.05). 2, MTT experiments showed not the intervention group IDO-HepG2 cells HepG2 cells of T cells against the cytotoxic activity was significantly lower than the empty plasmid group and blank control group, the measured value (35.00 ± 2.20)%, (74.51 ± 1.44)%, (75.30 ± 1.67)%, the former and the latter two were statistically significant (P lt; 0.05); while 1-MT intervention, T cells against cytotoxic activity of HepG2 cells were higher than intervention groups, namely, (71.33 ± 2.10)%, (81.27 ± 2.42)%, (80.91 ± 1.95)%, there was significant difference before and after intervention (P lt; 0.05). 3, flow cytometry analysis of the reaction system mixing ratio of Treg cells, not in the intervention group IDO-HepG2 cell group was significantly higher than the proportion of Treg cells transfected with empty plasmid group and blank control group, ie, (10.53 ± 1.05)%, ( 4.03 ± 0.17)%, (3.34 ± 0.18)%, the former and the latter two were statistically significant (P lt; 0.05); 1-MT were added after the intervention, Treg cells were lower than the proportion of 1-MT did not interfere with group, the value of (6.37 ± 0.15)%, (1.43 ± 0.10)%, (1.57 ± 0.13)%, there was significant difference before and after intervention (P lt; 0.05). 4, a mouse model of human liver cancer, flow cytometric analysis of Treg cells in peripheral blood of mice proportion of high expression of IDO in tumor-bearing mice was significantly higher than the proportion of Treg cells transfected with empty plasmid group and the control group, namely (15.33 ± 1.18)%, (8.96 ± 0.53)%, (8.14 ± 0.57)%, the former with the latter two were statistically significant (P lt; 0.05). Conclusions 1, IDO possible by increasing the ratio of regulatory T cells to inhibit cancer cells (HepG2 cells) and T cells of the immune apoptotic function toxicity. 1-MT inhibit this effect of IDO. 2, the in vivo experiments confirmed overexpression of IDO can increase the proportion of Treg cells.