| Background and Purpose:Macrophages play an important role in antitumor immunity. First, macrophages are immune surveillance cells. They can kill and remove senescent and malignant cells. Second, macrophages are important antigen presenting cells, they can present antigen to T lymphocytes. Third, macrophages are crucial effector cells. They can remove tumor cells directly. Activated macrophages can release many kinds of cytokines, IL-K IL-12 TNF-a and NO etc, which play an important role in macrophages ' antitumor activities. IFN- γ is the key stimulator of macrophages. Activated NK and T cells secrete it. Besides activating macrophages , IFN- Y also have other antitumor activities. It can promote APC to express MHC-II and tumor cells to express MHC-I, so It can enhance the recoganization between CTL and tumor cells, APC and T cells. IFN- γ also benefit the development of Thi and inhibit the development of Th2.IFN- γ can promote the expression of TNF- α ligand of tumor cells, so it can improve the sensitivity of tumor cells to TNF- α . Preview work have shown that macrophages activated by IFN- γ in vitro have antitumor effect. In this experiment , Mouse IFN-γ expression vector is constructed and antitumor effects of mouse peritoneal macrophages transfected with IFN- Y is observe in vivo and in vitro.Methods:1. Construction of mouse IFN- Y expression vector pcDNA3.1-IFN- γ Mouse spleen cells were separated and cultivated in 10% RPM1640 culturemedium (100 n g /ml PHA 10%F C S) for 4 hours. Then spleen cells were collected and total RNA was separated with Sangon kit K361. The IFN- γ mRNA was amplified by RT-PCR and nested PCR. The cDNA was purified by QIAGEN gel extract kit and liganded with pGEM-T vector. Through α -complementation and restriction analysis screening, The recombinant vector pGEM-IFN- γ was selected . The recombinant vector pGEM-IFN- γ was amplified and extract from JM109 bacteria. The purified pGEM-IFN- γ Plasmid was cut by restriction endonuclease BamHI and Hindlll. The cDNA of IFN- Y was collected and liganded with BamHI Hindlll digested pcDNA3.1 plasmid fragment. The recombinant mouse pcDNA3.1-IFN- γ was screened by restriction analysis and sequenced in Sangon company.2. Separation and culture of mouse peritoneal macrophagesMouse was killed and immerged into 70% ethanol for 10 minutes. 3ml ice-chilled PBSH( lOU/ml glycogen, 10% FCS ) was injected into mouse peritoneal . peritoneal liquid was drawn back 5 minutes later. Washing and collecting peritoneal macrophages by centrifugation. Cells was cultivated in 5 ml RPMI1640 under 37'C, 5% CO2 condition .3. Antitumor effects of mouse peritoneal macrophages activated by supernatant of IFN - Y gene transfected macrophage in vitroMouse peritoneal macrophages were transfected with recorabinant vector pcDNA3.1-IFN- γ . After transfecting 24 hours, the transfecting liquid was removed. New culture mediums was added to cells and continue cultivating for 48 h. Setting up pcDNAS.l plasmid and Calcium phosphate transfecting group .The culture medium was collected and stored at ?20. Collecting another group of peritoneal macrophages and cultivating them in 96-well flat-bottom microphates. Respectively adding culture medium from pcDNA3.1-IFN- γ -. pcDNAS.l -. Calcium phosphate transfecting groups and RPMI1640 to macrophages , after lOh cultivating , the culture medium was removed and add another l00ul RPMI1640 containing 1 X 104 H22 cells . RPMI1640 containing H22 cells were collected 48h later and the result was measured by MTT assay.After 48h transfection ,the transfected macrophages were collected and their total RNA was extract by Sangon kit 361 , The expression of INF- γ was measured by RT-PCR and PCR.4. IFN- Y gene transfect peritoneal macrophages in vivoPreparing Calcium phosphate transfecting liquid 7 days after Hi2 cells were incubated in mouse, injecting 500ul (containing 20ug pcDNA3.1-IFN- plasimd) Calcium phosphate transfecting liquid in mouse peritone...
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