Dissertation > Excellent graduate degree dissertation topics show

M2 pyruvate kinase (PKM2) related research

Author: ShiJingFei
Tutor: HeWei；CuiLianXian
School: Beijing Union Medical College
Course: Immunology
Keywords: TCRγδ PKM2 γδT cells monoclonal antibody preparation ELISA kit exploration
CLC: R737.31
Type: Master's thesis
Year: 2011
Downloads: 292
Quote: 0
Read: Download Dissertation

Abstract


So far only a few of ligands recgnized byγδT cell receptor (TCRγδ) were identified. In recent years, using TCRδchain complementarity determining region 3 (CDR3δ) peptide binding assay system, our laboratory successfully discovered and identified a number of TCRγδnew ligand, which laid the structural basis for comprehensive clarification of the function ofγδT cell.In the pre-research of our group, M2-type pyruvate kinase (PKM2) was caught by OT3 peptide (a dominant sequence of CDR3δfrom ovarian cancerγδTIL) as a probe from protein extracts of the ovarian cancer. And PKM2 had high levels of expression in tumor cells. The first part of this study, we adopt various methods to reserch PKM2 binding properties, cellular localization and its stimulation role for activatingγδT cells, in order to determine whether a new ligand of TCRγδ.By Western blot, the binding of OT3 peptide with PKM2 was analyzed; with Western blot and immunohistochemistry, the expression of PKM2 in tumor cells and tissues was observed;by flow cytometry and confocal method, PKM2’s location in the tumor cell was tested. The test of immobilized PKM2 expandingγδT cells in vitro and the detection of IFN-γsecreted by PKM2-stimulatedγδT cells were used to observe the function of PKM2.The results show that: The binding of PKM2 with OT3 peptide and containing a large number of PKM2 in tumor cells hints that the catching of PKM2 by OT3 peptide from total protein of tumor cells extracts is reasonable and that finding OT3 binding proteins by this method is feasible;PKM2 is widely expressed in tumor cells and tissues, but not expressed on the tumor cell membrane; PKM2 neither expandsγδT cells nor stimulates the secretion of IFN-γfromγδT cells in vitro. Visible, PKM2 is not expressed on the surface of tumor cells, and can’t stimulate activation ofγδT cells. PKM2 does not have properties of TCRγδligand. However, the results of immunohistochemistry showed that PKM2 in renal cell carcinoma, colorectal and lung cancer was highly expressed, suggesting that PKM2 as a possible cancer biomarker had the clinical value.The second part of our work is to prepare PKM2’s monoclonal antibodies, in order to provide necessary reagents for verifying PKM2 as a tumor biomaker.We took recombination Human PKM2 as immunogen, and BALB/C mice were vaccinated three times; by ELISA assay serum antibody titers of the mice were tested; the mouse whith highest titer was selected for the impactive immunization; 3 days later the blood of the nouse was collected as the positive control of following experiments; the spleen cells of the mouse were prepared for cell fusion with the SP20; the fusion hybridomas were cultured in methyl cellulose semi-solid culture medium; by ELISA, positive clones were identified; by Western blot, we identify their specificity; Rrotein G affinity chromatography was used to purify monoclonal antibodies; then purified monoclonal wre conjugated with HRP; explore the preparation of the ELISA kit for PKM2’s quantitative detection. By the methods above, we successfully prepared the monoclonal antibodies of PKM2; three of them wre available for Western blot experiment; two antibodies (5-G10 and the 4-D10) were successful purified and labeled with the HPR; and then using these two mAbs, we conducted a sandwich ELISA for PKM2, whose results showed there was positive binding reaction. This work provides a foundation for the following development of the ELISA kit for PKM2’s quantitative detection.

Related Dissertations

  1. Phenotypic and Functional Study of NK and γδT Cells in Malignant Pleural Effusion,R730.4
  2. The Cross-talking between Decidual γδ T Cells and Trophoblasts in Human Early Pregnancy,R339.22
  3. Determination of γδT Cells and Total T Lymphocytes in Lung Cancer Patients,R734.2
  4. The Genotypic and Phenotypic Identification of MULT1/MULT1S Transgenic Mouse & the Recognition Mechanism of γδT Cells to Lipid A,R392
  5. The Establishment of Expanding Gammadelta T Cells in Vitro, the Isolation of i-IELs and Immunofluorescence Techniques,R392
  6. Difference of Resistance to Salmonella Enterica Infection among Allelic Variations in TLR4 in Chickens,S858.31
  7. Research of the Effect of Tuberculin Pure Protein Derivative on ΓΔT Cell,R96
  8. γδT cells the research of on the the the adjust the the the role of of the of the the 's immune of the lung macrophages,R392.1
  9. The Expression and Clinical Application ofαβ/γδ/δ1/δ2 T Cells and Cxcr4/Ccr5 in Peripheral Blood and Tumor Tissue of Human Breast Cancer,R737.9
  10. Preparation and Identification of Monoclonal Antibody Against Human Gastric Cancer Carcinoma,R735.2
  11. The Establishment of ELISA Method about Porcine Reproductive and Respiratory Syndrome Virus Recombinant Protein N and Preparation for Monoclonal Ntiboody,S852.65
  12. Study on Allergic Pulmonary Inflammation of CXCR3 Knockout Mice Sensitized and Challenged with House Dust Mite Extracts,R562.25
  13. Cyclosprine A Inhibits Breast Cancer Proliferation by Downregulating Expression Level of Pyruvate Kinase Subtype M2,R737.9
  14. M2 pyruvate kinase in human thyroid cancer cell proliferation role in the process,R736.1
  15. The γδT cells regulatory induced in vitro and ex vivo expansion of γδT chemoattractant preliminary study of the mechanism of,R392
  16. Cloning, Expression of F5 Fimbril Gene and Praparation of Its Monoclonal Antibody,S852.61
  17. Preliminary Studies on the Immuno-regulatory Phenotype of Murine γδT Cells,R392
  18. Preparation and Application of the Monoclonal Antibody Against Rabies Virus,S852.65
  19. Changes of γδT Cells in Blood of Rats with Osteosarcoma after High Intensity Focused Ultrasound Treatment and a Possible Mechanism,R738.1
  20. Clonality Analysis of γδT Cell in Patients with Pure Red Cell Aplasia,R556

CLC: > Medicine, health > Oncology > Genitourinary tumors > Female genital tumors > Ovarian tumors
© 2012 www.DissertationTopic.Net  Mobile