Dissertation > Excellent graduate degree dissertation topics show

The Development of Neovasculature Endothelial Cells Targeted Recombinant Mutant Human Tumor Necrosis Factor Alpha

Author: WangHui
Tutor: ZhangYingQi
School: Fourth Military Medical University
Course: Biochemistry and Molecular Biology
Keywords: biotechnology-derived drugs tumor necrosis factor alpha RGD peptide cytotoxocicity synergistic effect tumor inhibition tumor vasculature
CLC: R73-36
Type: PhD thesis
Year: 2004
Downloads: 142
Quote: 0
Read: Download Dissertation


Tumor necrosis factor alpha (TNFa) is a macrophage-derived cytokine originally identified for its ability to induce hemorrhagic necrosis of transplanted solid tumors in mice and for its cytotoxic activity against some tumor cell lines in vitro. But the clinical use of nativeTNF is mainly limited to local treatment, e.g. in TIM/ILP and the TNFerade transfer, because of its dose-limiting systematic toxicity. In these settings, the antitumor activity of TNF depends on indirect mechanisms associated with selective obstruction and damage of tumor-associated vessels rather than having toxic effects directly on tumor cells.RGD motif -containing peptide is a core sequence which mediates the binding of many ECM proteins to integrins, in which included the avB3 integrin. And by in vivo biopanning of phage random peptide library, phages carrying CDCRGDCFC peptide is found to accumulate in the tumor neovasculature. To date, this high affinity characteristic of RGD-containing peptide to the avB3 integrin has been widely employed to targeted deliver the radioelements, virus vector to the tumor neovasculature.To overcome the problems of systematic toxicity in cancer therapy, many TNF a mutant have been developed, among which rmhTNF, with a 7-amino acid deletion at N-terminal and 4-piontsubstitution at 8, 9, 10, 157 amino acid, has a higher anti-tumor efficacy and lower systematic toxicity as proved in preclinical and clinical investigation. And rmhTNF has been approved by SFDA to be used in combination with chemotherapeutics in later period lung cancer patients. To further improve the therapeutic index of rmhTNF, we fused CDCRGDCFC peptide to the N-terminal of rmhTNF, and hope that the resulting fusion protein (abbreviated to RGD4C-rmhTNF) could selectively act on the tumor neovasculature ECs and alter the barrier function of endothelium when administrated systematically. To our knowledge, this is the first description of fusion protein containing RGD4C peptide and rmhTNF.I .The laboratory-scale research of recombinant protein RGD4C-rmhTNFFirstly, fusion gene encoding recombinant protein RGD4C-rmhTNF was constructed and the RGD4C-rmhTNF protein was expressed in E.coli DH5 aby temperature shift, mainly in a soluble form. The expressed protein could bind to the anti-TNF Mab specifically. Then the engineered bacteria was cultured by 5L automatic control ferment pot, the cytotoxicity of cell lysate was 5x1022IU/L. The RGD4C-rmhTNF protein was purified to homogeneity by ammonium sulfate precipitation, anion and cation exchange chromatography. The purity was over 95% and the specific cytotoxicity reached 2 x108IU/mg. We also validated that the purified protein existed as a mix of monomer, dimmer and trimer.II Quality control and large-scale production of RGD4C-rmhTNF proteinTo meet the requirement of pre-clinical investigation, the production and purification procedure of RGD4C-rmhTNF protein were scaled up to manufacturing level. Two batches RGD4C-rmhTNF protein were purified to homogeneity and the products amounted to 93.92mg and 115.7mg respectively. Thebioactivity recovery rates were 91.43% and 95.59% respectively. Quality control requirement of the RGD4C-rmhTNF protein for tumor therapy was established. Highly purified recombinant protein undertaken quality control foe identity, purity, cytotoxicity and impurity residue prior to its use.III The effect of RGD4C-rmhTNF administrated alone or in combination with chemotherapeutics on the tumor cell in vivo and in vitro and a preliminary investigation of its mechanism.In this part, we show that the both RGD4C-rmhTNF and rmhTNF had no effect in decreasing the S-180 tumor burden when used alone. When in combination with ADM, the RGD4C-rmhTNF could significantly enhance the antitumor effects of ADM at doses ranging from 10000IU/kg ~250000IU/kg in S-180 sarcoma animal models and H22 tumor models, whereas the rmhTNF had no such synergistic effects. And we also found that the route of administration of RGD4C-rmhTNF, e.g.

Related Dissertations

  1. Effect of Reinforcing Materials on the Properties of Nitrile Butadiene Rubber,TQ333.7
  2. Expression of TNF-α, MMP-9 and VEGF in BALF of Rats with Emphysema and Their Mutual Relationship,R563.3
  3. Restructuring lampreys RGD peptides antithrombotic Pharmacodynamics,R285.5
  4. Research on Implicit and Explicit Instruction in Senior High School English Classes,G633.41
  5. Study of the Effects of CD59, CD55 on Lipid Rafts-mediated Singnal Transduction of T Cell,R392.12
  6. The Expression of TNF-α on Retinal Excitotoxicity Injury Induced by NMDA,R774.1
  7. High Fat Diet Induced Insulin Resistance and the Influence to Interleukin 6, Tumor Necrosis Factor a and P-IKBα,R587.1
  8. The Experimental Study about Protective Effects of Puerarin on Liver of Brain-dead Rats,R285
  9. Studies on Polysilicic Acid-polyferric Flocculant for Papermaking Wastewater Treatment,X793
  10. Study on the Corrosion-Preventing Efficiency and Synergistic Action of PhAMT,TG174.42
  11. Enzymatic Transesterification for Biodiesel Production from Chinese Kernel Oil,TE667
  12. Research on the Synergistic Effect of Photocatalyst between Phosphor and Titania,O643.36
  13. The Experimental Research of Thetriptolide and Hydrocortisone Anti-inflammatory Effects,R96
  14. Construction and Expression of Fusion Gene (RGD)3 - DAB391 Prokaryotic Expression Vector and Study of Its Tumor Depression Effect in Vitro,R737.9
  15. Effects of A-SAA on the Expressions of Inflammatory Cytokines mRNA , Protein and Related Kinases of 3T3-L1 Adipocytes,R587.1
  16. The Synergy Benefit Evaluation of Steel Enterprises Mergers and Acquisition in China,F426.31;F224
  17. The Research on Safety Competence Model for Rock Blaster in Coal Mine,TD235.41
  18. Dynamic Study on Co-gasification of Coal Char and Biomass Char,TQ541
  19. Involvement of Src-suppressed C Kinase Substrate in Experimental Autoimmune Encephalomyelitis,R744.51
  20. Effect of Leflunomide on the Transplanted Endometriosis Lesions in SD Rat,R711.71
  21. Peroxisome Proliferators Activated Receptor γ and Sterol Regulatory Element Binding Protein-1c in Nonalcoholic Fatty Liver Rat Liver Tissue and Significance,R575.5

CLC: > Medicine, health > Oncology > Oncology experimental study > Treatment of experimental
© 2012 www.DissertationTopic.Net  Mobile