Dissertation > Excellent graduate degree dissertation topics show

The Study of Dnazyme in Inhibiting Mutant p53 Gene Expression in Vitro

Author: FangPing
Tutor: JiangLiPing
School: Chongqing Medical University
Course: Pediatrics
Keywords: DNAzyme mutant p53 HT29 cell line
CLC: R346
Type: Master's thesis
Year: 2005
Downloads: 43
Quote: 0
Read: Download Dissertation

Abstract


Background and objective The formation and progression of tumor result from the imbalancebetween cellular proliferation, cell differentiation and apotosis which is dueto activation of oncogenes, inactivation of tumor suppressor genes, orvariation of genes which inhibit apoptosis. As “guardian of genome”, thep53 protein is an important player in this process, above 50% of all humantumors produce aberrant p53 protein. Mutant p53 (mp53) may actdominant-negatively by hetero-oligomerization, via the C-terminaltetramerization domain, with the wide type p53 (wp53) expressed from theremaining allele. The dominace of a mutant is rarely complete and,consequently, the wide-type allele is often inactivated in the course oftumor progression. Apart from neutralizing wp53, some mutants show awide spectrum of pro-tumorigenic effects in cells that lack wp53. Forinstance, mp53 may increase the tumorigenicity of cell, regulate gene,inhibit differentiation, increase mutation frequency or metastatic potentialand cause genomic instability in a wide-type p53-null background. Manyresearches focused on transfection of wp53 into tumor cell leading tosuppress tumor cell proliferation. However, there are no effective inclinical trials while the results in vitro are exciting. So it is an importantapproach in tumor gene therapy to eliminate the expression of mp53 thatwas overexpressed in tumor cell. Recently, there are many studies focusedon regulating the expression of mp53 with antisense oligonucleotides orribozyme. Deoxyribozyme (DNAzyme, DZ) is a novel approach which is beingdeveloped to specifically cleave RNA molecules at almost any targetedsequence. Based on RNA-cleaving function, DZ is derived from acombinatorial library of sequences by in vitro selection. The mostprominent DZ named “10-23” DZ consists of a catalytic core of 15nucleotides and two flanking arms of 4-15 nucleotides on either side. TheRNA substrate is cleaved at a particular location between an unpairedpurine (A or G) and a paired pyrimidine residue (C or U). It is highlysequence-specific to cleave almost any target RNA. From the first exampleof a DZ that could cleave RNA, biologists have built DZs for wide-rangingapplications in suppressing the expression of a variety of target genes inviral infectious diseases, tumor and cardiovascular diseases. However, DZhas not been studied for regulating the expression of mutant p53. We designed three DZs targeting mp53 (R273H) mRNA nearbymutant site. Their catalytic activities were analyzed by cell free assay, toscan a DZ which cleaving the mp53 mRNA specially. The catalyticactivities of many DZs supressing the expresion of mutant p53 mRNA andprotein were analyzed in HT29 cell. The overall objective of this study wasto explore the possibility for p53 gene therapy by using DNAzyme. Methods 1. Based on Satoro’s reports, information about p53 mutant inhttp://www-p53.iarc.fr/index.html and the secondary sturcture of p53mRNA predicted by RNAstructure and RnaViz computer program, wedesigned “10-23”DZ targeting the mutant p53 (R273H) mRNA. 2. The catalytic activities of DZs in cell free system: 345bp cDNAs ofwp53 gene from A549 and mp53 gene from HT29 were generated byRT-PCR. The amplified fragments were inserted into pBluescript II KS+,generated two recombinant plasmids, wp53pBs and mp53pBs. The RNAfragments of wp53 and mp53 which were the structures of “10-23”DZ weretranscribed in vitro using T7 polymerase. The RNA cleavage activities ofDZ were measured in cell free system. 3 The effects of DZs were tested in HT29 cell: DZs or ASOs weretransfected into HT29 cells, RT-PCR was performed for evaluating theinhibitory activity of DZs or ASOs on mp53 mRNA expression,immunocytochemical methods and Image Pro Plus 4.5 were used to detectthe p53 protein expression on HT29 cell. MTT assay were performed todetect the cell activity of HT 29. Results 1. The design and synthesis of DZs: Three “10-23”DNAzymesp53DZ7, p53DZ9, p53DZ11 having an arm with different nucleotudenumber in 3’ structure-binding arm were designed. DZs cl

Related Dissertations

  1. PITX1 and mutant P53 in gastric cancer and precancerous lesions and its clinical pathological significance,R735.2
  2. The Signicance of HER-2, p53 and Ki-67 Expression in Colorectal Cancer,R735.3
  3. Novel Aptamer-based Techniques for the Chemiluminescent Biosensors,TP212.3
  4. Detection Method of Lead Base on Nucleic Acid Fluorsence Probe,TQ075.1
  5. Identification on the Targeting Organ of Precancerous Lesion in LMP1-mtp53 Transgenic Mice and on the Interfering Effect of Qi-Boosting Toxin-Resolving Formulae on the Lesion,R739.63
  6. Expressions of COX-2 Protein and Mutable p53 Protein in Breast Cancer and Their Clinical Significance,R737.9
  7. Investigation of Functional Nucleic Acids in Photochemical Biosensor,TP212.2
  8. Studies of Functional Nucleic Acid-based Electrochemical Biosensors,TP212.3
  9. The Expressions of Mutant P53 and Fas/Fas-L in Rats Renal Tissue Induced by Paraquat and the Therapeutical Effects of Hepatocyte Growth-promoting Factors,R595.4
  10. Application of Nanoprobes for Detection of Trace Lead,R114
  11. Exper Study the Significance of Mutant P53、 P170 in Salivary Adenoid Cystic Carcinoma(SACC),R739.8
  12. Preparation and biological analysis of semiconductor nanomaterials,TB383.1
  13. Studies on the Reversal of Multidrug Resistance of Cancer by p53 Gene Therapy Combined with Radiotherapy,R73-3
  14. Expression and Correlationship of Apoptosis-inducing Factor(AIF) and Mutant p53(mp53) Protein in Gastric Cancer and Gastric Precancerous Lesions,R735.2
  15. The Clinical Experimental Research of Targeting EBV-LMP1 Deoxyribozyme Enhancing the Sensitivity of NPC Radiotherapy,R739.63
  16. MR Perfusion Imaging Evaluation Targeting EBV-LMP1 mRNA Deoxidation Ribozyme-assisted Radiotherapy Nasopharyngeal Carcinoma’s Clinical Study,R739.63
  17. Expression of mtP53 and HSP70 in Epithelial Ovarian Carcinoma and Clinical Value,R737.31
  18. A Simple, Rapid Assay for Kinetic Constants of RNA-cleaving Deoxyribozymes under Simulated Physiological Conditions,Q55
  19. The Studies on the RNA-DNAzyme,Q55
  20. Pilot Study of Nucleic Acid Antibiotics Targeted to Bacterial 16S rRNA,R392

CLC: > Medicine, health > Basic Medical > Human biochemistry, molecular biology
© 2012 www.DissertationTopic.Net  Mobile