| Laryngeal carcinoma is a common head and neck cancer, which main pathological type is squamous cell carcinoma. In recent years, the incidence of laryngeal squamous cell carcinoma increased significantly. Smoking, alcohol consumption, environmental factors, exposure to radiation, viral infections and lack of trace elements will cause laryngeal squamous cancer through variety of channels, combining effect of multiple molecular factors.At present, the main treatments of laryngeal cancer are surgery and radiation therapy, others comprehensive measures include chemotherapy, immunotherapy, gene therapy, Chinese medicine treatment, et al. The effective treatment should include:the removal of the total tumor, retaining of the function of the larynx, improving survival and the life quality of patient. In recent years, with the improvement of the diagnosis and treatment level all over the world, electronic laryngoscope and early localized biopsy can improve the survival rate of patients with laryngeal cancer. Even so, there are still many patients with poor prognosis, surgery recurrence and distant metastases. Therefore, searching for a safe, effective, side effects of treatment is a difficult task.The greatest feature of cancer cells is out of control of normal growth-control system. They can continue to divide and proliferate with the "immortality." Therefore, the focus of cancer treatment is to inhibit the proliferation and promote their death. Tumors often will be caused by mutation, phosphorylation or inactivation of multiple tumor suppressor genes. The normal tumor suppressor gene can be transferred into tumor cells and replace the function of the loss gene, which may achieve reverse the malignant phenotype, inhibit tumor growth, or even eliminate tumor, and may became a hot issue in tumor gene therapy. P16 gene is an important tumor suppressor gene in the human body, mutations and deletions in this gene is closely related with a variety of tumors, including head and neck cancer. An important way to study the anti-tumor gene therapy is to restore the wild-type p16 protein function. P16 protein is a inhibitor of cyclin-dependent protein kinase, either directly or indirectly regulate the cell cycle, and can induce cell cycle arresting in G1 phase. It also has a role in promoting tumor cell apoptosis. Therefore, we will study the impact of p16 protein in laryngeal carcinoma cells and explore from apoptosis and cell cycle.Natural killer cells are an important part of the innate immune system. They may not be pre-sensitized when they recognize and kill target cells, so they are the body's first line of defense in anti-tumor immune defense. Since the cytotoxic activity of NK cells is regulated by cell surface receptors and cytokine, anti-tumor effects of NK cells in the immune system is very important. Meanwhile, the tumor cells can modulate or inhibit NK cell's function through the expression of immunosuppressive factors or other negative surface ligand. Thus regulate the body's anti-tumor immune response. At last it can result in a immune escape environment.In recent years, there is evidence that the p16 gene introduced into the tumor cell will invoke the changes of surface molecule expression and cytokine secretion in tumor cells. This may be involved in the formation of immune escape of tumor cells. Therefore, we will investigate the relationship between p16 protein and NK cell's antitumor effect. This may provide more theoretical basis for the applications of p16 protein as a tumor therapeutic target.Research purposes1. To Amplify of recombinant adenovirus vector carrying wild-type p16 gene and introduce the recombinant adenovirus vector into Hep-2 cells, and then to observe the expression of p16 protein in Hep-2 cells.2. To investigate the proliferation inhibition of wild-type p16 protein on laryngeal carcinoma Hep-2 cells and to explore its mechanism from apoptosis and cell cycle.3. To explore the effect of wild-type p16 on the sensitivity of laryngeal carcinoma Hep-2 cells to NK cells and observe the regulation of wild-type p16 on the NK cell cytotoxic activity through Hep-2 cells.Research methodsPart I:The amplification of recombinant adenoviral vector in HEK-293 cells and the observation the expression of p16 protein in Hep-2 cells.We use human embryonic kidney cell line HEK-293 to amplify the recombinant adeno viral vector with wild-type p16 gene or LacZ and use the plaque assay to determine the titers of adenovirus. After recombinant adenoviruses infect the hep-2 cells, we will use flow cytometry and western blot to detect the expression of p16 protein in the Hep-2 cells.Part II:Study the wild-type p16 protein on the growth of laryngeal carcinoma Hep-2 cells in vitro and in vivo.The proliferation of Hep-2 cell infected with Ad-pl6 or Ad-LacZ was detected of by CCK8. Effect of p16 protein on Hep-2 cell apoptosis and cell cycle will be tested by flow cytometry. The morphology of apoptotic cells will be observed by Electron microscopic. the expression of apoptosis protein and cell cycling protein will be detected by Q-PCR. The Hep-2 cells were inoculated subcutaneously in nude mice, and the recombinant adenovirus or PBS will be injected intratumorally. Then we will observe the volume of tumor. Tunnel method will be used to detect tumor tissue apoptosis.Part III:Study the effect of wild-type p16 on the sensitivity of Hep-2 cells to NK cells and the regulation of wild-type p16 on the NK cell cytotoxic activity through Hep-2 cells.After infected Hep-2 cell, we will observe the effect of wild-type p16 on the sensitivity of Hep-2 cells to NK cells by CCK8, the expression of NK receptor ligands on the surface of Hep-2 cells by flow cytometry, the gene expressions of IL-6, IFN-y, IL-10 and TGF-? by qRT-PCR and the secretion of TGF-? in the Hep-2 supernatant by ELISA. After incubating NKL cells with Hep-2 cell culture supernatant, the killing activity of NKL on Hep-2 cell will be observed by CCK8 method. By adding or blocking TGF-? mode, we detect the effect of TGF-P in the Hep-2 cell culture supernatant on the regulation of NK cell function.Research resultPart I:The amplification of recombinant adenoviral vector in HEK-293 cells and the observation the expression of p16 protein in Hep-2 cells.1. We found the virus titers of Ad-p16 and Ad-LacZ were respectively 6.5×1010 and 4.8×1010.2. Flow cytometry analysis showed that the expression of p16 protein in Hep-2 cells were respectively 29.5%,85.0%,92.7% and 95.4% at 3h,6h,12h and 24h after the Hep-2 cells infected with Ad-p16 group, while the expression of p16 protein in the Hep-2 cells of Ad-LacZ group and uninfected group had no p16 protein expression.3. The results of Western blot test also showed the expression of p16 protein in Ad-p16-infected Hep-2 was significantly increased.Part ?:Study the wild-type p16 protein in laryngeal carcinoma Hep-2 cells in vivo Growth.1. At 24h,48h,72h,96h after the Hep-2 cells were infected with Ad-p16, the proliferation inhibition rates of Hep-2 cells were respectively 3.003%±0.497 (24h), 12.031%±1.946 (48h),24.857%±1.473 (72h) and 30.35%±1.916 (96h), and compared with Ad-LacZ, there were significant differences;2. The cell apoptosis was detected by flow cytometry, the apoptosis rate of Hep-2 cell in Ad-p16 group changed from 9.99%(24h) to 26.98%(48h),37.87%(72h). Compared with Ad-LacZ group, there was no significant difference at 24h, while there were statistically significant difference at 48h and 72h.3. The electron microscopy revealed that Ad-pl6-infected cells existed chromatin condensation and obvious apoptotic bodies while The Ad-LacZ-infected Hep-2 cells rarely existed chromatin condensation and apoptotic bodies.4. In Ad-pl6 group, the rate of Hep-2 cells in G0/G1 phase increased significantly (48.36%), the S phase cells significantly reduced (38.26%), while the G2/M phase cells cell had no change, Compared with Ad-LacZ group.5. Compared with Ad-LacZ group, the pro-apoptotic genes expressions of p53, Caspase-8 and Bak in Ad-p16 group were up-regulated while the anti-apoptotic gene Survivin and Bcl-2 were down-regulated, there were statistically significant difference. But there was no significant difference in the expression of Bcl-xl;6. Compared with Ad-LacZ group, the gene expression of cell cycle proteins p21 and cyclinDl in Ad-pl6 group were increased, there were statistically significant differences, but there was no significant difference in the gene expressions of E2F2 and CDK4.7. The tumor growth in the mice of PBS group and Ad-LacZ group were rapid, and there was a certain linear relationship between the tumor volume and time in these two groups. Compared with Ad-LacZ group, tumor volume in Ad-p16 treatment group there was no statistical difference before 12 days, after 16 days the growth in Ad-p16 group change slowly, and there were significant difference, which showed the tumor growth regression trend appears.8. Tunnel staining showed the Tunnel percentage of positive cells in untreated group was 30.56%±1.842, in Ad-LacZ group was 34.44%±2.572, and in Ad-p16 group was 71.78%±3.244. The apoptosis of tumor tissue was significantly high in Ad -p16 group, compared with Ad-LacZ group.Part ?:Study the effect of wild-type p16 on the sensitivity of Hep-2 cells to NK cells and the regulation of wild-type p16 on the NK cell cytotoxic activity through Hep-2 cells.1. The cytotoxic activity of NK cells against Hep-2 cells of Ad-p16 group improved and had significant difference, compared to Ad-LacZ group.2. Flow cytometry showed the expression of MICA/B and ULBP2 on the surface of Hep-2 cells in Ad-p16 group were raised. Compared with Ad-LacZ, there had significant differences.3. qRT-PCR test results showed that the gene expressions of IL-10 and TGF-? in Hep-2 cells of Ad-p16 group down-regulated while the gene expressions of IL-6 and IFN-y up-regulated. Compared with Ad-LacZ group, all had a statistically significant difference.4. ELISA test results showed the concentration in the culture supernatant of TGF-P from Ad-pl6 group is lower than the other two groups, and had a statistically significant difference.5. We used the culture supernatants of Hep-2 from different group to incubate NKL cells, and then detected the cytotoxic activity of NKL according to the effect/target ratio of 4:1 ratio. The results showed the killing efficiency of NKL from untreated group was 44.80%±1.277, Ad-LacZ treated group was 44.43%±1.241, there was no significant difference. The killing efficiency of NKL from Ad-p16 group was 77.10% ±1.850, compared with Ad-LacZ group, p<0.0001.6. We add anti-TGF-? Ab to the culture supernatant from Ad-LacZ group to block TGF-? action and showed that the NK cell cytotoxicity changed from 44.43%±1.241 to 58.33%±1.525, and there was a statistically significant. We supplement TGF-? protein to Ad-p16 group and showed that the killing activity of NK cells decreased from 77.10%±1.850 to 58.73%±2.554, there was a significant difference.Conclusion1. Recombinant adenovirus Ad-p16 have the capable of making Hep-2 cells express p16 protein, so Ad-p16 can be a good vector to provide a good foundation for the next step study.2. The wild-type p16 protein expression in Hep-2 can inhibit the growth of Hep-2 cells in vitro and in vivo, the mechanism has two aspects, one is to induce apoptosis of Hep-2 cells; the second is to make Hep-2 cells arrested in G0/G1 phase.3. The wild-type p16 protein expression in Hep-2 can up-regulate the expression of NKG2D ligands on Hep-2 cells and increase the sensitivity of Hep-2 cells to NK cells; on the other hand, The wild-type p16 protein can alter the expression of cytokines in Hep-2 cell, such as TGF-? and IL-10 decreased, IFN-y increased, which can enhance the killing ability of NK cell.SignificanceForm this subject, we reveal that in the mechanism of tumor inhibition of p16 protein have two different pathway:one is to inhibit tumor cell growth, the other is to promote killing ability of NK cell on tumor cells. At present, there are few scholars to study the relationship between these two approaches, and we only have carried on the preliminary discussion to them. But I believe that with the development of immunology and gene therapy, the mechanisms of tumor suppressor gene will be more in-depth understanding. It will create a new road for tumor immunotherapy. |
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