| BackgroundHepatocellular carcinoma is a high-risk cancer in our country. Many datas suggest:the rate of hepatocellular carcinoma is the third in malignant tumor, which is known as the king of cancer. About110000people die of hepatocellular carcinoma each year, accounting for45%of the world hepatocellular carcinoma deaths. Because of eating aflatoxin B and carrying HBV surface antigen, there is a high incidence of primary hepatocellular carcinoma in Guangxi. At present, the therapy of hepatocellular carcinoma most depends on radiotherapy, chemotherapy and surgery at home or abroad. But traditional treatments have many disadvantages:significantly toxicity, recurrence and high price. It is urgently demanded to find more effective methods to overcome these disadvantages. In recent years, immunological cytotherapy might be expected. People hope that chemotherapy can unite biological immunotherapy, which can completely control recurrence of hepatocellular carcinoma and cure hepatocellular carcinoma.Immunological cytotherapy is a new method in cancer therapy. Many researches showed that immunocyte played a significant role in the tumor immunological cytotherapy. As one of lymphocytes, natural killer cells have becomed a hot spot since natural killer cells were found in1975, which are believed to be the main effects to kill cancer cells. Natural killer cells account for about10-15percentage in peripheral blood. NK cells are an important part of nonspecific immune system which don’t even need to be stimulated, can kill and destroy the cells infected by virus and tumor cells, and no toxic reaction to normal tissue. NK cells drive its function through inhibitory receptors or express cytokines. Activated function of NK cells is associated with Fas-L, granzyme, TNF-L, TRAIL and so on.Newcastle disease virus7793(NDV7793) was isolated from wild duck of PoYang lake in Jiangxi, which belongs to Avulavirus genera of the Paramyxoviridae family, it is highly pathogenic to birds, but it can kill tumor cells and rarely damage normal cells after infecting human, which suggests that NDV7793can be used as a potential anti-cancer agent. Our researches have found that NDV7793can selectively kill pharyngeal carcinoma, hepatocellular carcinoma and colon cancer, and the killing activity is related with dosage and incubation time of virus. NDV7793can activate the macrophages of the mouse, and the killing effect of the mouse macrophages is enhanced after NDV7793stimulation. Based on these inspiration, HepG2is treated as target cells in this topic, which are susceptible to NK cells, the killing effect on hepatocellular carcinoma by NDV-stimulated NK cells was investigated, preliminary explored the killing effect whether is related to TRAIL, TNF-α and IFN-γ or not. All above, revealing the mechanism of NDV7793can promote NK cells to kill HepG2in vitro.ObjectivesTo reveal the mechanism of NDV7793can promote NK cells to kill HepG2in vitro.MethodsNK cells were purified by the immunomagnetic micro-beads, and NK cells were activated by NDV7793in vitro, then mixed culture NK cells and hepatocellular carcinoma, the killing effect of NK cells was analyzed by LDH. RT-PCR was used to detect the expression of TRAIL mRNA after NDV7793stimulated NK cells. TRAIL protein was tested by flow cytometry (FCM), TNF-α and IFN-γ were detected by ELISA. At last, we used IFN-γ antibody to block IFN-γ, and then detected the expression of TRAIL by FCM, at the same time, we set up PBS, IL-2, IFN-γ negative and positive control. Experiments were performed to ascertain whether the killing effect of NK cells is related to TRAIL, TNF-α and IFN-γ or not, and to confirm whether the signal pathways of TRAIL is involved in IFN-γ or not.Results1. NK cells were purified by the immunomagnetic micro-beads, the purity of NK cells can reach to90.60%±1.15%;2. NDV7793can enhance NK cells to kill HepG2; the killing effect was related with virus dosage and incubation time;3. NDV7793could enhance the expression of TRAIL mRNA of NK cells, its expression was correlated with virus dosage and incubation time, the expression of TRAIL mRNA reached to summit when NK cells were stimulated4h by NDV7793;4. FCM results:the expression of TRAIL of NK cells could reach summit (11.97%±0.503%) when NK cells were activated16h by25.6Hu NDV7793at different time; In addition, its expression could reach summit (22.77%±1.8%) when NK cells were activated16h by102.4Hu NDV7793;5. NDV7793could promote NK cells to excrete TNF-α. Its expression could reach to summit (0.130±0.03) when NK cells were activated by different titers of NDV7793; its expression could reach summit (0.184±0.01) when NK cells were activated12h by204.8Hu NDV7793;6. NDV7793could enhance the excretion of IFN-γ of NK cells. At different times, its expression could reach summit (2.296±0.157) when NK cells were activated16h by25.6Hu NDV7793; NK cells were activated by different titers of NDV7793, IFN-γ could reach to summit (2.875±0.108) when NK cells were activated by25.6Hu NDV7793;7. Neutralizing test of IFN-γ indicated:the expression of TRAIL was decreased when NK cells coated with IFN-γ antibodies.Conclusions1. NDV7793can enhance NK cells to kill HepG2; the killing effect is related to the excretion of TNF-α, IFN-γ and the expression of TRAIL protein;2. The expression of TRAIL may have other pathways besides the signal pathway of IFN-γ. |
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