| Objective Laryngeal cancer is a common malignant tumor in Otorhinolaryngology, of which 90% are squamous cell carcinoma. At present, the incidence of laryngeal cancer is increasing obviously. It is not only because of the improvement of clinical diagnostic technique, average life augment, but also the aggravation of air pollution and smoking. Statistics found that: the phenomena of the higher incidence of laryngeal cancer in cities than in countryside, in heavy industry cities than in light industry cities. Surgery as the most commonly treatment for laryngeal cancer has been used a hundred years. Recently, with the development of head and neck surgery,the laryngeal surgery has been improved obviously and the 5-year survival rate and quality of life of patients with laryngeal cancer improved significantly.But there are still 30% -40% of patients will relapse or metastasis after radical operation and chemoradiation. In this situation most of these patients were not sensitive to chemoradiation and the 3-year survival rate of them was barely 10%. Improved the curative effect of laryngeal cancer has already been an urgent task in this situation. With the development of molecular biology and the genetic engineering, the gene immune therapy have received increasing attention and showed a great prospect.Interleukin 24 (IL-24) is also called melanoma differentiation associated antigen-7(MDA-7).It is a secreted protein which belongs to IL-10 cytokine family and mainly expressed by CD3~+ T lymphocytes and monocytes. Studies have shown that IL-24 was the only one which could not only inhabit tumor growth and neovascularization but also a cytokine could stimulate immune system. However this inhibition is not independent of p53, Rb, p16 and other tumor suppressor genes. Also, it has no effect on normal cells. Now, it has become the hot spot of cancer therapy. Although mda-7/IL-24 can work through many different ways, its mechanism is not clear yet. There is no report update whether it can act on Laryngeal cancer. We cloned its cDNA, overexpressed it and analyzed its role on Laryngeal cancer cell line.Methods1.Mda-7/IL-24 cDNA was cloned from PHA treated human peripheral blood lymphocytes with RT-PCR and construct plasmid PEasy-T1 Simple-IL-24。2.Construction of Ad.mda-7/IL-24: Amplified mda-7/IL-24 gene product from the plasmid PEasy-T1 Simple-IL-24 with PCR and inserted into the pAd-Track-CMV shuttle vector. The resultant plasmid was cotransfected into E.coli BJ5183 cells with pAdEasy plasmid to undergo homologous recombination. Then the linearized recombinant plasmid was transfected into 293 cells and their titers were measured by TCID50 method. The correct recombinant adenvirus Ad.mda-7/IL-24 and Ad were identified by PCR and Western-blot.3.Ad.mda-7/IL-24 infect laryngocarcinoma cell and detect related gene expression. Ad.mda-7/IL-24 and Ad infect laryngocarcinoma cell Hep2,tetrazolium salt (MTT) method to observe the effection on proliferation of Hep2. The related gene and protein expression were detected by RT-PCR and Western-blot.Results1. Mda-7/IL-24 cDNA was cloned from PHA treated human peripheral blood lymphocytes with RT-PCR and construct plasmid PEasy-T1 Simple-IL-24. It was confirmed by DNA sequencing and digesting.2. Vectors related to adenovirus expression including shuttle plasmid pAdTrack-CMV-mda-7/IL-24, the adenovirus vector containing the target gene and the empty adenovirus vector pAd were obtained by recombinant DNA technology. These vectors were constructed successfully approved by sequence analysis and electrophoresis. The linearized recombinant plasmid was transfected into 293 cells by lipofectamine method. The green fluorescence could be seen in transfected 293A cells under fluorescence microscope. RT-PCR detected IL-24 mRNA expression and Western blot showed that IL-24 protein expressed in transfected cells.3. After transfected with IL-24, the proliferation effect of Hep2 cells was decreased and the cytotoxicity to Hep2 cells increased significantly. Transcription levels of Fas,BAX,Caspase-3 showed remarkable increased but the BCL-2 showed decreased.ConclusionsThe main purpose of tumor therapy is to inhibit tumor growth and improve patient prognosis,the primary task of basic research is to clarify the mechanism of cancer generation and the tumor suppressor gene function. Our research reveal the overexpression of IL-24 on the role of laryngeal cancer cells and its mechanism of action by cloning mda-7/IL-24 and construct recombinant adenovirus packaging of adenovirus containing the gene。The results showed that the adenoviral vector express IL-24 gene was successfully constructed, and the virus containing the IL-24 was also successful expressed. It is proved that IL-24 had visible inhibition in the proliferation of the Hep2 laryngeal cancer cells. This inhibition of proliferation may be by changing the proportion of apoptotic genes and anti-apoptotic genes to achieve. This work has provided technical basis for gene therapy with mda-7/IL-24 on Laryngeal carcinomas and added some new idea to the mechanism of carcinogenesis.
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