Construction Of The Plasmid And Expression Of CD28 And 4-1BB In K562 Cells

BackgroundNasopharyngeal carcinoma is one of the most common malignant tumor in southeast Asia, especially of the southern provinces in China. At present the first choiced method for nasopharyngeal carcinoma is radiotherapy treatment, the 5-year survival rate of comprehensive treatment is 50% - 60%, but even after formal and eradication therapy, the local recurrence and distant metastasis are still the main causes of mortality in the middle-late NPC patients. The effect of comprehensive treatment again is not ideal, but side-effect increased significantly, the quality of patients'life decline meanwhile. Therefore, it is necessary to provide a safe and effective second-line treatment for the middle-late nasopharyngeal carcinoma patients. Biological therapy has become the fourth-largest treatment except surgery, chemotherapy and radiotherapy. Immunotherapy is an important branch of the biological therapy: non-specific immunotherapy (the autologous peripheral blood CIK cell therapy technology) are widely used in clinical already, with efficiency around 30%, it may increase with improving the technology; Specific immunotherapy is better than therapeutic non-specific immunity treatment which has killer effect for a certain tumor cells.EB virus is a external pathogenic factor related to the existence and genesis of various tumors, it was confirmed that the EB viral infection have closely relation with nasopharyngeal carcinoma. The existence of the virus provides a potential site for cytotoxic T lymphocytes (CTL), many researchs about EB virus associated tumor immunotherapy are aimed at its expression of the antigen LMP2.But this specific immunal therapy needs to culture a large amount and efficient specific CTL in vitro, than infuse them into human'body after the processing, the traditional method of PBMC/OKT-3 has the problem of disabilities in specificity anti-tumor immune response with amount, activity and stability. CTL are defective, one of the main reasons is insufficient or defective of expression of T cells costimulatory molecules. CD28 and 4-1BB are two of its most important molecules on the T cells have been discovered, the former express in static T cells, induced CD4 + T cell activity priority, and the latter express in activate T cells, induced CD8+ T activity priority. with antigen respectively cell activity, they combinate B7(CD80) and 4-1BBL in APC respectively, and collaborat MHC-Ag signals to improve the immunogenicity of tumor cells, They promote the activation, proliferation, differentiation and apoptosis of T cells, and play an important role in the continuous activation procession.Although these total collaborative stimulate pathways mediated by costimulatory moleculars are non-specific, they are indispensable for the T cell's activation by mutual adjustment and complementing each other in the process of mediating immune responses. So it is a basis of amplification system of CTL in vitro to look for an experiment method of expressing these two kinds of costimulatory moleculars stably. It also provides conditions of immunotherapy for nasopharyngeal carcinoma and other tumors.Objective:Synthesis the sequence of gene of human CD28 and 4-1BB molecules, integrate them into the eukaryotic carrier, and express them on the surface of leukemia cells (K562). Methods:1. Cultivating people leukemia cells (K562).2. Synthesis the sequence of gene of CD28 molecules, cloning them in pcDNA3.1-Neo plasmid, and transfecting them in e.coli DH-5α.3. Synthesis the sequence of gene of 4-1BB molecules, cloning them in pcDNA3.1-Neo plasmid, and transfecting them in e.coli DH-5α.4. Amplificating and extracting the reorganizative plasmids from e.coli DH-5α.5. Transfecting K562 cells by reorganizative plasmids.6. Screening the transfected K562 cells.7. Flow cytometric analysis of the expression of CD28 and 4-1BB.Results:1. Synthesis the sequence of gene of CD28 molecules, and it confirmed to be coding sequence of CD28 by DNA sequence analysis.2. Getting the reorganizative plasmid by cloning the synthetic products in pcDNA3.1-Neo plasmids, and electrophoresis result reveals that it includes the gene fragments we need.3. Synthesis the sequence of gene of 4-1BB molecules, and it confirmed to be coding sequence of 4-1BB by DNA sequence analysis.4. Getting the reorganizative plasmid by cloning the synthetic products in pcDNA3.1-Neo plasmids, and electrophoresis result reveals that it includes the gene fragments we need.5. Flow cytometric analysis shows that 29.54% transfected K562 cell express CD28 molecular.6. Flow cytometric analysis shows that 28.31% transfected K562 cells express 4-1BB molecular. Conclusions: This research shows that using such experiment method can successfully clone and stablly express stimulate molecules of CD28 and 4-1BB on leukemia cell K562, and it can be used as experimental basis of CTL amplification system in vitro.