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Effects of Differentiation-inducing Reagents and Gene Transfection of Heterologous Human DNA Polymerase β on Cell Biological Characteristcs

Author: JinGe
Tutor: DongZiMing;WuJingLan
School: Zhengzhou University
Course: Pathology and Pathophysiology
Keywords: DNA polymerase beta Esophageal cancer Induced differentiation Reverse transcription - polymerase chain reaction Western blot Base excision repair
CLC: R73-3
Type: PhD thesis
Year: 2003
Downloads: 66
Quote: 0
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Abstract


Tumor pathogenesis people more and more aware of the importance of DNA repair enzymes in the maintenance of cell genome stability role, DNA repair enzymes abnormal relationship with tumor growing concern by the people, the current has become a hot topic in the etiology of cancer research. The base excision repair enzyme (base excision repair, BER) is one of the major DNA repair enzymes. Caused by various endogenous and exogenous factors. Genomic DNA of 2-6 nucleotides injury and abasic sites (apurinic / apyrimidinic, AP), it is able to clear the maintenance of genomic stability. DNA polymerase β (polymerase β, pol β) is one of the key enzymes in a single base excision repair. The pol β widely exist in eukaryotic and prokaryotic. Minimum molecular weight found similar enzyme is highly conserved among different species. Activity is very low and does not vary with the cell cycle removing involved in nucleotide gap fill short segments (single), other than to maintain the stability of the genome may participate in DNA replication, recombination, and cancer drug resistance of some drugs, and its mutations throughout the cell cycle and abnormal expression may have a tumor promoter role. Many researchers have found in a variety of tumor tissues and cell lines DNA pol β gene mutations, high expression of mammalian cell DNA pol β can induce chromosomal instability, increased risk of cancer. However pol β mutations and expression and genome stability relations as well as the study of the role in carcinogenesis is still in its infancy, still need further study. Establish pol β stably transfected cell lines and applied research is an effective way. Pol β has established stable transfected cell lines rarely, and similar foreign research focuses pol β gene in mice, and human pol β gene stable transfected cell lines has not been reported. In this study, the different types of DNA polymerase beta eukaryotic expression vector transfected mammalian cells, preliminary observation of the gene expression of wild-type and mutant DNA polymerase beta cell biological characteristics, Zhengzhou University doctoral dissertation in 2004 biological effects of cell differentiation inducers and different types of human DNA polymerase p gene transfection to lay the foundation for further study the relationship of the repair defects caused by mutations in the DNA polymerase p and tumor development. Tumorigenesis is the result of cell proliferation and differentiation disorders, the regulation of cell proliferation and differentiation is controlled by multiple genes in a multi-link complex process, affected by a variety of factors. Induced differentiation of malignant tumor cells in vitro and in vivo differentiation inducer role reversal towards the direction of the normal cells to differentiate. Induction of differentiation and treatment of cancer as a new way of cancer treatment and research is very active in recent years. In the process of induction of differentiation with cancer is closely related oncogenes and tumor suppressor genes can change in which wild-type p53 (wild tyPe p53, pair p53) plays a key role. We apply two differentiation inducer effect on others to esophageal Eca109 cell lines, observed changes in expression and function of DNA polymerase in cell differentiation phenotype p. The study is divided into two parts: 1. Differentiation inducer all-trans retinoic acid (all.transretinoic acid, ATRA), dimethyl sulfoxide (d and e Imperial sulfoxide, DMSO) of Eca-109 cells induced to differentiate and the relationship between the DNA polymerase p; 2 different types of polymerase p eukaryotic expression vectors were stably transfected into mammalian cell line, identification and cell biological characteristics; experimental methods: 1. the establishment of all-trans retinoic acid (ATRA) and dimethyl sulfoxide (D Mso) induced Eca-109 cell line and PcDNA3 .1 a lp a PM3 PcDNA3.1 of Yifan polp stable transfection MH3T3 cell lines, while building cell lines of the control groups. Trypan blue exclusion dye test and MTT to record cell growth curve. 3 the. The application random primers preparation Biotin labeled the the Bio YiFan p53 cDNA probe. The in vitro transcription machine polp DNA single enzyme the linear template, plus SP6RNA polymerase, rNTPs and Bi. A 11-dUTP was prepared by in vitro transcription of the antisense RNA probe, the labeled probe to DNA dot blot detection sensitivity the carried polp situ hybridization. 4. The positioning observed expression signals relative quantification: oil microscope observe cells in situ hybridization signal and immunoreactivity (POLB IR, PCNA TR) positioning and its strong and weak levels of integral value. At the same time without labeled probe and specific antibodies specimens as a negative control in situ hybridization and immunohistochemistry. 5 Total cellular RNA was extracted, reverse transcription polymerase chain reaction (Rr-PCR) assay po Wei mRNA expression. 6 soft agar colony formation assay determination of induced differentiation of colony formation rate plus drug group and the control group, the biological effects of cell inhibition rate of Zhengzhou University, 2004 PhD thesis differentiation-inducing agents, and different types of people the DNA polymerase day gene transfection . 7 application of DNA methyl acid denaturation the Green faction Browning GS were counted cell proliferation / differentiation of the cells and the ratio. Flow cytometry differentiation group and cell cycle of the transfected and control groups. 9 compared to immunoblotting POLB protein expression level of cells in each group. 10 nuclear protein extract induced differentiation in each group and the control group, detection BER functionality. Results: differentiation-inducing effect of differentiation-inducing agents Eca-109 cells and its relationship with the between polp expression and function. Cell growth curve: ATRA group (5 pm. Ratio) and DMSO (l .5 %) cells compared with the control group, the growth slowed down to four days the difference was significant (p lt; 0.05). Cheng p53 gene upregulation 2. Angeles p53 gene expression: application the 5 p moUL eight TRA and 1.5% DMSO induced Eca-109 cells tend to be divided, not dosing group difference was significant (p lt; 01) Of 3.DNA polp gene mRNA, protein expression: ATRA and DMso, in gene and protein levels reduce polp expression. 4.ATRA and DMSO of Eca-109 cells in soft agar set off the formation of ability: of ATRA and DMSO are significant with the inhibition of Eca-109 cells in soft agar to form colonies capacity, does not drug group compared differences have a significant (p- lt; 0 .01). ATRA the inhibition rate of 89%, DMso inhibition rate of 92%. Colony not only control group than the medication group colony large, and the number of cells in a single colony more. 5. Proliferating cells ratio count: Compared with the control group, eight T

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