Inhibition Of The Liver Cancer Transplantation Tumor In The Mice By NDV7793

Background and Object:Biological immunotherapy become a hot topic in cancer therapy because of trauma to the patient and have toxic side effects, high efficiency, multiple lesions or metastatic malignant equally effective, long-term advantages of good anti-cancer effect. Study found that the virus antitumor effect not only kill the tumor specialty, but also be able to activate the host immune system for tumor immune response, so the virus anti-tumor therapy as a new biological immunotherapy. So far NDV as a single-stranded negative RNA of paramyxovirus, not only to specifically kill tumor cells direct, but also stimulation the host immune system to produce anti-tumor effect is very significant. Our group use NDV attenuated strains of duck isolated from Poyang Lake in Jiangxi province. (Named WDK/J/7793/2004, called NDV 7793) for anti-tumor research.We found NDV7793 not only can activate NK cells in vitro,and CD4^T cells, but also in a variety of ways, including the way of TRAIL in vivo enhance NK cell activity against hepatocellular carcinoma cells.We want to reserch the effect of NDV7793 inhibitory subcutaneous transplantation tumor of liver cancer in mice.Exploring the NDV7793 effect immune function for tumor infiltrating lymphocytes (Tumor-infiltrating lymphocytes, TIL) in subcutaneous transplantation tumor of liver cancer tumors for the purpose of NDV7793 as a biological immunotherapeutic agents used in clinical practice to provide experimental evidence.Methods1. The inhibition of NDV7793 on the liver cancer transplantation tumorH22 cells were cultured, about 1×106 single mouse hepatoma H22 cells were injected into the right armpit of mice to establish mouse liver cancer subcutaneous transplantation tumor model. After the success of the subcutaneous transplantation tumor model of liver cancer in mice, given NDV7793 tumor injection after amplification and purification, continuous dosing days respectively,1 d and 2 d,3 d,4 d,5 d,6 d and 7 d, the control group with PBS instead of NDV. Based on its survival and intratumoral CD4+CD25+CD127low regulatory T cells (Treg) in TIL, and CD8+T cell ratio choose NDV7793 the best dosing days.80 tumor-bearing mice which were established the Liver cancer subcutaneous transplantation tumor model were randomly divided into PBS negative control group, IL-2 positive control group, influenza virus (FLU) in the control group, NDV7793 administration group. Each group were 20, intratumoral injection,0.1 mL per mice, from the beginning of the modeling success administered continuously for 4 days,10 mice in each group were randomly selected for each group of mice survival analysis, the remaining mice were sacrificed 24h after stopping, stripping tumor weighed to calculate the inhibition rate.2. The influence of NDV7793 on the TIL immunity in the liver cancer transplantation tumor.After grinding sterile tumor tissue stripping acquisition by Percoll gradient centrifugation TIL, TIL was divided into three parts:(1) Take 2* 106 TIL cells were treated with the antibody after incubation, the flow cytometry CD4+CD25+CD127lowTreg, CD8+T cell ratio of total TIL cells; (2) TIL cells in cell culture tubes plus a complete medium, and cultured after 16h, the supernatant was collected by ELISA assay IL-2, IFN-y, concentrations of TNF-a; (3)TIL as effector cells and CFSE labeled rat hepatoma H22, Hepal-6 target cells by 5:1 effector to target mixing ratio, culture 4h, added PI, flow cytometry CFSE+PI+cell death that is the percentage of the target cells.Results1. The inhibition of NDV7793 on the liver cancer transplantation tumor1) Successfully established the liver cancer transplantation tumor model, the successfulrate reached 97.5%(78/80).2) Analysis of NDV7793 survival time of mice at different times of administration can be obtained:Survival of NDV7793 group of tumor-bearing mice with the increase in the number of days NDV7793 administration change, and survival of tumor-bearing mice PBS negative control group will not be changes; NDV7793 administration of tumor-bearing mice longest survival time of 4d.3) After the analysis of the survival of the administration group can be obtained:NDV7793 administration group survival was significantly longer than the IL-2 positive control group and PBS negative control group and FLU administration control group, the difference was statistically significant.4) Analysis of the inhibitory effect of each group after administration can be obtained:NDV7793 treatment group tumor weighter than PBS negative control group, IL-2 positive control group, FLU administration control group, the difference was statistically significant; NDV7793 administration group of H22 inhibition rate was significantly higher than PBS negative control group, IL-2 positive control group, FLU administration control group, the difference was statistically significant.2. The influence of NDV7793 on the TIL immunity in the liver cancer transplantation tumor.1) Analysis of mice at different times within the NDV7793 isolated from tumor-bearing mice tumor TIL of CD4+CD25+CD127lowTreg, CD8+T cells in the proportion of total TIL available:NDV treatment group CD4+CD25+CD127l0W Treg in the total TIL percentage is higher than PBS negative control group lower, CD8+T cells in the total TIL percentage higher than PBS negative control group, the difference was statistically significant;NDV7793 administration Id to 4d, NDV7793 administration days and CD4+CD25+CD127low Treg in the total TIL percentage was negatively correlated with that as NDV7793 increase the number of days of administration, CD4+CD25+CD127low Treg but decreased the percentage of the total TIL, CD8+T with increasing percentage of total TIL,NDV7793 administration in mouse tumor 4d of CD4+CD25+CD127low Treg in the total TIL lowest percentage (2.07±0.36)%, CD8+T in the total TIL highest percentage (20.04±1.44)%, but NDV7793 administered to the first paragraph 4d to 7d, NDV7793 administration days and CD4+CD25+CD127lowTreg in the total percentage of TIL was positively correlated with the increase of the number of days NDV7793 administration, CD4+CD25+CD127!owTreg TIL percentage increases,CD8+ T in the total TIL percentage is decreased.2) Analysis of the administration group isolated from tumor-bearing mice tumor TIL of CD4+CD25+CD127low Treg, CD8+T cells in the proportion of total TIL available:NDV-administered group TIL of CD4+CD25+CD127low Treg is less than PBS negative control group, IL-2 positive control group and FLU administration control group, the difference was statistically significant; NDV administration group CD8+T cells in the proportion of total TIL higher than PBS negative control group, FLU administration group, the difference was statistically significant, but NDV7793 TIL administration group of CD8+ T compared to the total percentage of TIL and IL-2 positive control group, the difference was not statistically significant.3) Analysis of the administration group isolated from tumor-bearing mice tumor TIL cells in cell culture supernatant IL-2, IFN-y, TNF-a concentration results can be obtained:NDV treatment group TIL cell culture supernatant IL-2, IFN-y, TNF-a the average concentrations higher than PBS negative control group, IL-2 positive control group, and the FLU administration control group, the difference was statistically significant.4) Analysis of the administration group isolated from.tumor-bearing mice tumor TIL killing rate of mouse H22, Hepal-6 hepatoma cell lines available:NDV treatment group TIL killing rate of rat hepatoma cell line H22, Hepal-6 in PBS negative the control group, IL-2 positive control group and FLU administration control group, the difference was statistically significant.Conclusions1. NDV7793 can inhibit the growth of mouse liver tumor, liver tumor survival NDV7793 mice can be prolonged, and different NDV7793 time of administration, prolong survival varies.2. NDV7793 can change the mouse liver tumor intratumoral TIL of CD4+CD25+CD127l0WTreg, CD8+T in the total proportion of TIL, and mouse liver tumor in the tumor TIL of CD4+CD25+CD127low Treg, CD8+T cells with different proportions of total TIL administration time of change, while still raised NDV7793 TIL isolated from rat liver cancer cells within the tumor xenograft culture supernatant IL-2, IFN-y, TNF-a concentrations of level, while NDV7793 can significantly improve the isolated from rat liver tumor in the tumor TIL in vitro cytotoxicity of murine H22, Hepal-6 hepatoma cell lines.