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Preparation and Identification of a Novel Particle Vaccine Based on TEM-8 and Investigation of Their Anti-tumour Efficiary

Author: LiuPing
Tutor: LiangHouJie
School: Third Military Medical University
Course: Oncology
Keywords: Tumor TEM-8 CTL Immunotherapy
CLC: R392
Type: Master's thesis
Year: 2010
Downloads: 36
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Abstract

Background and purpose: the growth and metastasis of solid tumors is dependent on angiogenesis and, therefore, anti-angiogenesis has also become an effective tool of anti-cancer therapy. Currently generated VEGF as a target for passive immunization of anti-angiogenic drugs in clinical made some progress, the ensuing anti-angiogenic therapy has also been in-depth, in order to activate cytotoxic T lymphocytes (cytotoxic T lymphocyte, CTL) mainly active immunotherapy strategy played an important role in the comprehensive treatment of malignant tumors. Active immunotherapy can induce their own tumor-specific immune response, not only can recognize and eliminate tumor cells, but also can induce immunological memory, and to prevent recurrence of the tumor compared with other conventional treatment methods. Continuously administered to the patient brought trouble and a severe economic burden can be reduced. Therefore, the use of anti-angiogenic vaccine prospects are very attractive. So far, anti-angiogenic molecules vaccine treatment, the target antigen primarily VEGFR-2, VEGF, FGFR, MMP-2 molecule, these molecules are not only rich in tumor tissue, and in some normal tissues higher expression levels similar target for the design of molecular vaccines potentially harmful itself. 2000 Croix found nine specific genes highly expressed in colon cancer vascular endothelial tumor endothelial marker 1 to 9 (tumor of endothelial markers were named as TEMs). TEM-8 mRNA in adult mice, are not expressed in normal tissue or trace expression detected by in situ hybridization, in its tumor endothelial expression is very abundant. , TEM-8mRNA and TEM-8 protein in the human body in almost all colon cancer, esophageal cancer, bladder cancer, lung cancer cases high expression, but not in the next in the same case cancer and normal tissues expression in the corpus luteum and wound TEM-8 protein molecules of new blood vessels in the healing process is not detected. These findings suggest that the TEM-8 is the best one of the targets of immunotherapy candidate. In the the molecular vaccine research later confirmed by TEM-8, a separate the molecules vaccine immunogenicity lower future can effectively stimulate an immune response, and in combination with other antigens, later scholars introduction of DC vaccines in clinical effective TEM-8 increase the immunogenicity of anti-tumor effect, but there is the transfer efficiency is not high, the operation inconvenient, and higher cost. Cationic peptide DNA transporters (cationic peptides DNA delivery systems) is one of the rapid progress in the field. This class of DNA delivery carrier for some rich in positively charged amino acids of the polypeptide, such as poly-L-lysine (ploylysine, [K] n), which can be electrically and enriched with the negative charge (phosphate groups) plasmid DNA polymerization, compression, so that the diameter of about several hundred nanometers, loose plasmid DNA polyacetal become dense particles of several tens of nanometers, so that the purpose of easy to be the intake of eukaryotic cells so as to achieve gene transfection. Build \The purpose of this project is to assess the effectiveness and side effects after the cationic CPPs transformation TEM-8-based molecular vaccine after the anti-tumor immune therapy, in order to evaluate whether it is necessary to immunotherapy TEM-8 research. Methods: We first constructed cation fusion peptide [K] 16-tat49-57, fusion peptides by reverse phase high pressure liquid chromatography identified its purity, was identified by mass spectrometry and its molecular weight, in particular NaCl concentration, with the rich in negative charge pTEM-8 eukaryotic expression plasmid combined to build a new particle vaccine particle size scanning transmission electron microscopy, TEM-8 protein expression westernblot detection; 2. construct CT-26 colon cancer model, based TEM -8 new particle vaccine Balb / c mice, the ability of anti-tumor vaccine research. Immunized mice once a week for three consecutive times. The survival time of the mice were observed and changes in tumor volume, tumor blood vessel growth using immunohistochemical observation and calculation of tumor microvessel density; 3. Confirm the anti-tumor effect of the vaccine, we further study the anti-tumor mechanism of vaccine toxicity; Results: 1. synthetic polypeptides identified by HPLC and the purity has reached 96.57% by mass spectrometry analysis of the molecular weight of 3390.44 Da, and successfully constructed particulate vaccine, TEM and analysis software, its uniform particle size diameter of less than 25nm, found vaccine requirements and its transfection and immunodetection MW 63KD expected results; 2. experimental results show that, based on the TEM-8 particles of the tumor volume of the vaccine group and control group significantly difference (p lt; 0.05), prolonged survival of tumor-bearing mice. Vascular endothelial cells within the tumor tissue with anti-CD31 monoclonal antibodies for immunohistochemistry results showed that TEM-8 vaccine group microvascular density (MVD) is significantly lower than the control group; 3. Stimulate CTL 51Cr release assay proved after vaccine immune mice were able to stimulate CTL immune response ELISPOT assay showed the effect of in vivo vaccine can effectively stimulate antigen-specific CTL, respectively, using anti-CD8, anti-CD4 or anti-CD8 monoclonal antibodies for protective immunity test and found the mice can not get protective effect after vaccination; anti-CD4, the tumor volume of the experimental group and the control group of mice there is still a significant difference (p LT; 0.05) to prompt the CD8 T cells play a major role in the anti-tumor; conclusions: 1 successfully constructed novel particle vaccine based on the TEM-8, while effectively mediated gene expression; particle vaccine based on the TEM-8 can effectively inhibit tumor growth rate, increase the survival of tumor-bearing animals; 3. CD8 T cells play a major role in the anti-tumor immune response induced by DNA vaccine;

Preparation and Identification of a Novel Particle Vaccine Based on TEM-8 and Investigation of Their Anti-tumour Efficiary

Abstract

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