Dissertation > Excellent graduate degree dissertation topics show

Exosomes Derived from Il-12-anchored Renal Cancer Cells Increase Induction of Specific Antitumor Response in Virto

Author: ZhangYao
Tutor: WuXiaoHou
School: Chongqing Medical University
Course: Urology
Keywords: Renal cell carcinoma exosome Immune-related proteins Glycosylated phosphatidylinositol Human interleukin- 12 Gene Eukaryotic expression plasmid Exosomes Interleukin- 12 Tumor vaccine
CLC: R737.11
Type: PhD thesis
Year: 2010
Downloads: 112
Quote: 0
Read: Download Dissertation

Abstract


Part I: Kidney Cancer the endogenous exosome extraction and identification purposes: Separation and Purification of kidney cancer cells secreted the exosome, detect the protein composition of exosome immune explore the potential clinical value. METHODS: Cultured human renal carcinoma cell line RC-2. Ultrafiltration and sucrose / heavy water density gradient ultracentrifugation separation and purification of the exosome, transmission electron microscopy to observe the morphology, Western blot was used to detect the composition of immune-related proteins. The immunocytochemistry kidney cancer kidney cancer-specific antigen G250 expression. Results: ultrafiltration and sucrose / heavy water density gradient ultracentrifugation, successfully isolated the exosome. TEM RCC derived exosome class round dish, by the lipid bilayer surrounding the hemisphere, with a diameter between 30 ~ 80nm. Western blot analysis: renal cell carcinoma-derived exosome expression kidney cancer-specific antigen G250, heat shock protein 70 the (Heat Shock Protein70, HSP70) and cell adhesion molecules (Intercellular Adhesion Molecule-1, ICAM-1). The immunocytochemistry showed: the human renal cancer RC-2 high expression in renal cell carcinoma-specific antigen G250. Conclusion: ultrafiltration and sucrose / heavy water density gradient ultracentrifugation can isolate purified renal cancer cells secrete the exosome to the exosome expression of immune-related proteins of HSP70, ICAM-1, its load kidney cancer-specific antigen G250, immunogenic and can be used Preparation of kidney cancer vaccine. The second part of human interleukin-12 gene with GPI signal peptide sequence of the fusion gene eukaryotic expression plasmid and identification purposes: a glycosylated phosphatidylinositol (Glycosylphosphatidylinositol, GPI) signal peptide sequence of human interleukin-12 gene spliced ??into fusion gene construct the fusion gene eukaryotic expression plasmid. Methods: The human interleukin-12 (IL-12) P35 亚基基因亚 clone to pBudCE4.1 plasmid, the recombinant plasmid pBudCE4.1/IL-12A. Human placental alkaline phosphatase -1 (hPLAP-1) C-terminal signal peptide sequence with the P40 subunit of the IL-12 gene splicing, the fusion gene was subcloned into the recombinant plasmid pBudCE4.1/IL-12A recombinant plasmid pBudCE4.1/IL-12A/IL-12B-GPI. Plasmid restriction analysis and sequencing. Results: Kpn Ⅰ and Xho Ⅰ the digestion pBudCE4.1/IL-12A plasmid can be cut approximately 762bp purpose of article with the correct sequencing. The recombinant plasmid pBudCE4.1/IL-12A/IL-12B-GPI with Kpn I, XhoI, BamHI and Xba Ⅰ digestion, can be cut approximately 762bp and 1104 two purposes strip, respectively, the chain of the IL-12A and IL- 12B-GPI. The sequencing results not gene mutation, and the correct nucleotide sequence and the comparison of the coding sequence of GenBank. . Conclusion: The success of the eukaryotic dual of expression plasmid pBudCE4.1/IL-12A/IL-12B-GPI the building, which laid the foundation for the further application of IL-12 anchor modified exosome. The third part of the IL-12 anchor Preparation and characterization of modified exosome kidney cancer vaccine? Objective: Preparation of IL-12 anchor given modified the exosome (the exosome-GPI-IL-12 EXO/IL-12) kidney cancer vaccine and its protein composition and functional identification. Methods: of glycosylated phosphatidylinositol signal peptide sequence with interleukin 12 gene fusion construct eukaryotic double expression plasmid pBudCE4.1/IL-12A/IL-12B-GPI of transfected renal cancer cells, obtained stably transfected cell lines. The laser scanning confocal microscope and a phosphatidylinositol-specific phospholipase C (PI-PLC) the purpose of flow cytometry eluted proteins (GPI-IL-12) expression in renal cancer. Density gradient centrifugation, ultrafiltration and sucrose / heavy water from stably transfected cell lines cultured the the supernatant prepared vaccine EXO/IL-12. Transmission electron microscopy to identify the form of the iconic molecular Western blot identification EXO/IL-12, of HSP70, ICAM-1 the composition, as well as kidney cancer-specific antigen G250 and target protein expression of GPI-IL-12. ELISA assay EXO/IL-12 load the amount of of IL-12. Of IL-12, IFN-γ release experiments and CFSE-labeled lymphocytes proliferation assay membrane binding function. Results: laser confocal microscope shows: renal carcinoma cells stably transfected with plant height target protein GPI-IL-12 expression. PI-PLC-eluting flow cytometry shows: IL-12 is a form of anchored proteins binding in renal cell carcinoma cell surface. Culture supernatants from stably transfected cell lines can be isolated and purified EXO/IL-12 vaccine for the class round dish, double membrane structure, diameter 30 ~~ 80 nm, high expression of HSP70, ICAM-1, G250 and GPI- IL-12. The ELISA assays 10μg of EXO/IL-12 containing the approximately 80 ± 9.6pg/ml of IL-12. 10μg EXO/IL-12 lymphocytes in vitro can induce the secretion of IFN-γ in an amount of 174.3 ± 14.6pg/ml, The exosome 40.7 ± 9.5pg/ml compared, a significant difference (P lt; 0.01). And in vitro, EXO/IL-12 can significantly induced lymphocyte proliferation, proliferation index was 1.45 ± 0.015, the exosome (1.29 ± 0.01) compared to the significant difference (P lt; 0.01). Conclusion the: pBudCE4.1/IL-12A/IL-12B-GPI plasmid stability transfected kidney cancer cells can preparing EXO/IL-12 vaccine, the vaccine expression of GPI-IL-12, load kidney cancer specific antigen The G250 can The Vitro significant induction of T lymphocyte proliferation and secretion of IFN-γ. The results show that: the membrane-bound IL-12 has a biological function, IL-12 is anchored the exosome modified significantly improve its immunogenicity. The fourth part of the IL-12 anchor modified renal cell carcinoma-derived exosome vaccine research purpose of the anti-tumor effects in vitro: EXO/IL-12 vaccine in vitro induced lymphocyte killing effect of renal cell carcinoma. Methods: isolated and cultured lymphocytes and dendritic cells, scanning electron microscopy the morphological identification of dendritic cells from the peripheral blood of healthy volunteers. Of dendritic cells loaded EXO/IL-12 stimulating factor and lymphocytes in vitro co-culture for 48 hours, to stimulate the activation of lymphocytes as effector cells, CFSE pre-marked renal cancer cells as target cells, a different effect - target than added, after a total of 48 hours of incubation, the CFSE / PI double labeled flow cytometric analysis of renal cancer cell killing effect, control PBS, EXO, IL-12. CFSE choose a different pre-labeled target cells (human renal cell carcinoma of human bladder cancer and colon cancer cells), flow cytometry CFSE / PI double labeled its destruction rate differences, explore EXO/IL-12 induced lymphocyte whether the killing effect of antigen-specific. Results: The results showed that: in effect - target ratio of 50:1, IL-12-induced activation of lymphocytes against renal cancer cell killing rate was 10.34 ± 3.92%, higher than the group with PBS 0.7 ± 0.26% (P lt ; 0.05); the EXO group induced activation of T lymphocytes of renal cell carcinoma killing rate was 24.3 ± 3.8%, significantly higher than the IL-12 group and PBS group, the difference was significant (P lt; 0.01). Most EXO/IL-12 induced lymphocyte killing of target cells, the destruction rate of 53.7 ± 3.9%, compared with the group, the difference was significant (P lt; 0.01). , EXO/IL-12 activated lymphocytes and renal cancer cell killing rate of RC-2 was significantly higher than that of the T-24 bladder cancer and colon cancer cell line SW480 (killing rate were 9.8 ± 2.5% and 10.8 ± 2.1% ), the difference was statistically significant (P lt; 0.01) Conclusion: EXO/IL-12 vaccine induced strong antigen-specific cytotoxicity of lymphocytes in vitro, is expected to become the new vaccine for the treatment of kidney cancer.

Related Dissertations

  1. Research and Implementation on Knowledge Representation of Gene Regulatory Network,Q78
  2. Research on Combinatorial Regulation of Multiple Transcription Factors,Q78
  3. Cloning and Expression of CHS and CHI Genes and Their Regulation on the Accumulation of Flavonoids in ’Cara Cara’ Navel Orange (Citrus Sinensis Osbeck) and ’Guoqing NO.4’ Satsuma Mandarin (Citrus Unshiu Marcow),S666.4
  4. Chemical Structures and Mechanism Research of Anti-aging Leading Compounds from Natural Products,R285.5
  5. CYP2C19 Gene Polymorphism in Patients with Ischemic Cerebrovascular,R743
  6. The Expression Survey of BMP Signalling Pathway in the Human Embryonic Tooth Germ,R78
  7. Cloning and Expression of Growth Hormone Gene in Cobia (Rachycentron Canadus),Q786
  8. Effects of Different Reconciled Corn Oils on Antioxidative Stress, Lipid Metabolism Enzymes, and Abundance of Immune-related Genes of Broilers,S831.5
  9. Polyethylenimine Conjugated Stearic Acid-g-chitosan Oligosaccharide Micelles for Antitumor Gene Therapy,R450
  10. An Approach for Identifying a Plant Resistance Gene Based on the Random Forest,Q943
  11. Functional Analysis of Proteins Encoded by RNA2 of Wheat Yellow Mosaic Virus,S435.121
  12. Comparative Study on Reproductive Biological Characteristics of Helicoverpa Armigera and Helicoverpa Assulta (Lepidoptera:Nuctuidae),S433
  13. Preliminary Study on the Biological Control Potential of the Hypovirus CHV1-CN280 and Functional Analysis of P29,S436.64
  14. The Detection of the Virulence-related Gene from Vibrio Alginolyticus and the Study on Preservation Methods,S943
  15. Establishment of the Molecular Identifying System of Curvualria and Application in Difficult Species,Q949.32
  16. Isolation and Identification of Infectious Bronchitis Virus in Henan and the Full Genome Sequence Analysis of Isolated Strains HN104 and HN091,S852.65
  17. Isolation, Identification and Molecular Characteristics of Japane Encephalitis Virus in Henan Province,S852.65
  18. Study on Synthesis and Physiological Function of N-carbamoyl-glutamate,R914
  19. Expression Dynamics of Pheromone Binding Proteins and Expression Influences by Mating and Knockdown of Cryl in Spodoptera Exigua,S433.4
  20. Molecular Cloning, Mrna Expression and Rnai of Nadph-Cytochrome P450 Reductase Gene in Helicoverpa Armigera (Hǘbner),S435.622.3
  21. Functional Analysis of tal (transcription activator-like) Genes of Xanthomonas Oryzae,S435.11

CLC: > Medicine, health > Oncology > Genitourinary tumors > Urinary tumors > Kidney,renal pelvis tumor
© 2012 www.DissertationTopic.Net  Mobile