The Construction And The Role In MHCⅡ Regulation Of Anadenovirus Vector Carrying CⅡTA Gene

Pancreatic carcinoma is a kind of high malignancy, rapid progress, poor prognosis of tumors. In recent years, the mobidity of pancreatic cancer has been increasing all over the world. Surgery offers the major treatment of pancreatic cancer. But most of the pancreatic cancer patients haven't the chance of radical surgery because the early clinical symptom of pancreatic cancer is atypical, or even association with distant metastasis. What's more, due to the resistance to cytotoxic drugs of pancreatic cancer cells, the chemotherapy have little effect. Therefore, it is urgent to search for the new effective treatments.In the process of tumor immunity, T cells play a key role in immune reaction. In most tumors, including pancreatic cancer, tumor cells can escape attacks of the immune system by reducing the expression of MHC I , MHC II, and the expression of costimulatory signals. So the tumor antigens can not be present to T cells effectively. The immune system of host was depressed, and could not recognize the tumor cells. Therefore, promoting the T cell activation is one of the keys of tumor immunotherapy.MHC II molecules mainly express on the surface of T cells, B cells, macrophages and APC. It is the necessary signals of T, B cell activation. The expression of MHC II molecules is mainly on the transcriptional level. Satoh A confirmed that it is a close relation between high expression of MHC II molecules and good prognosis of tumor. CIITA was first discovered in 1993. CIITA is the "molecular switch" in the expression of MHC II genes. It plays a key role on the constitutive and inducible expression of MHC II genes. It can determine the expression and the level of MHC II molecules. It is a good therapy target in the MHC II molecules related disease. Therefore, we can use CIITA to regulate the expression of MHCII molecules and then transform the normal and pathological cells. We can up-regulate the level of MHCII molecules through the exogenous stimulation for CIITA. And then, immune escape of tumor cells can be blocked after the presention of tumor antigen. This may be helpful to the treatment of tumor.The purpose of this study is to establish the adenovirus carring CIITA and confirm the promotion of MHC II molecules by CIITA transfecting in vitro. The expecting results of this study may be helpful in providing a new way in the immunotherapy of pancreatic cancer.First, the recombinant about adenovirus vector with CIITA. Objective: To construct the adenovirus vector that contains CIITA gene.METHODS: Recycled the PCR products with CIITA ,and inserted it into pUC57,use double digestion of EcoRI and XhoI and recycle the 3300bp target gene with U-Gene gel purification. Double digest the pShuttle-GFP-CMV vector with EcoRI and SalI, link the target gene and pShuttle-GFP-CMV by the T4DNA ligase . and then we can get the shuttle plasmid (pShuttle-GFP-CMV-CIITA),digested by Kpn I Single Double digest pShuttle-GFP-CMV-CIITA by I-Ceu I / I-Sce I, recycling fragment,link the pAdxsi vector and fragment ,get the adenovirus vector( pAdxsi-GFP-CIITA), Identify the vector by XhoI digestion and transfect into HEK293 cells that cultured virus. Amplified, purified recombinant AdCIITA, and virus titer.Results: The recombinant shuttle plasmid pShuttle-GFP-CMV-CIITA by Kpn I digestion ,and it fully consistent with the mouse CIITA (NM-007575) that published in GeneBank. The recombinant adenovirus AdCIITA that produced in HEK293 cells after the adenovirus pAdxsi-GFP-CIITA successfully constructed piay a pathogenic role to HEK293 cells. And purified by repeated infection, the virus titer equal to 2.0×1011PFU/ml.Conclusion: The recombinant adenovirus containing mouse CIITA gene has been successfully constructed.Second, MHC II molecules expression for test.Objective: To confirm the promotion about the constitutive and induced expression of MHC II molecules by transfection of Ad-CIITA in vitro.Methods: Determine the amount of virus through the gradient of infection Use relative cell number 1:1,10:1,100:1 virus, partly transfected P388D1 cells and Hela cells that stimulated by IFN-γ(500U / ml) for 48 hours in advance. Observed transfection efficiency under fluorescence microscope. Transfected P388D1 cells and Hela with Adenovirus Ad-CIITA and blank control.Collect the cells after24,48,72 h transfection, FACS analysis the expression of MHC II detected Molecule.Real Time PCR detect the expression of CIITA.Results: The gradient of the virus infection showed better transfection efficiency was 100:1. Real Time PCR showed comparing transfected cells with control,the expression of CIITA gene was significantly enhanced (p﹤0.05).FACS analysised that the expression of MHC II molecules in P388D1 cell has no significant difference before and after tasnsfection (p> 0.05); the expression of MHC II molecules in Hela cells has significant difference before and after tasnsfection with stimulating 48 hours with IFN-γ(500U/ml) in advance (p <0.05).Conclusion: Ad-CIITA can not promote the expression of MHC II molecules in macrophage expression cells,but can enhance the expression of MHC II molecules.in tumor cellsThird, biological changes with the human and mouse pancreatic cancer cells by Ad-CIITA transfection.Objective: Through the transfection of Ad-CIITA ,confirm the biological change in induced pancreatic cancer cells.Methods: Determine the amount of virus through the gradient of infection Use relative cell number 1:1,10:1,100:1 virus, partly transfected CaPanC-2 cells and PanC-02 cells that stimulated by IFN-γ(500U / ml) for 48 hours in advance. Observed transfection efficiency under fluorescence microscope. Transfected CaPanC-2 cells and PanC-02 cells with Adenovirus Ad-CIITA and blank control.Collect the cells after24,48,72 h transfection, FACS analysis the expression of MHC II detected Molecule.Real Time PCR and Western Blot detect the expression of CIITA.Results: The gradient of the virus infection showed better transfection efficiency was 100:1. FACS analysis that the PanC-02 cells and CaPanC-2 cells were induced expression . The expression of MHC II molecules significantly increased compared the transfection of recombinant adenovirus carrying CIITA with control,with stimulating cells 48 hours with IFN-γ(500U/ml) in advance (p <0.05). Real Time PCR showed comparing transfected cells with control,the expression of CIITA gene was significantly enhanced (p﹤0.05). Western Blot confirm the same thing from the protein.Conclusion:The transfection of Ad-CIITA can express CIITA gene and enhance the expression of MHC II molecules in CaPanC-2 cells and PanC-02 cells.Through this research, the subject has the following conclusions:1,The recombinant adenovirus containing mouse CIITA gene has been successfully constructed.2,Ad-CIITA can not promote the expression of MHC II molecules in macrophage expression cells,but can enhance the expression of MHC II molecules.in tumor cells3,The transfection of Ad-CIITA can express CIITA gene and enhance the expression of MHC II molecules in CaPanC-2 cells and PanC-02 cells. Summary: This study was prepared by recombinant adenovirus carrying CIITA in vitro transfection of P388D1 and Hela cells ,PanC-02 cells and CaPanC-2 cellswith the stimulation of IFN-γ(500U/ml), Initially confirmed the recombinant adenovirus carrying the CIITA transfected into tumor cells can be successfully expressed CIITA gene, and then enhance the expression of MHC II molecules in tumor cells .It provide a new way for the pancreatic tumor immunotherapy.