Construction And Application Of The Cell Strains With Down-regulated Expressed HOGG1 Genes Which Target To Oxidative DNA Damage And Repair

DNA damage and repair has always been a hotspot in toxicology and oncology. Among all kinds of DNA damages, oxidative DNA damage induced by free radicals is studied most popularly and is regarded as the most important factor in initiation and promotion of tumour. A great deal of studies showed that reactive oxygen species (ROS) can attack directly biomolecules such as DNA and induce DNA strand breakage or base modification. In all kinds of oxidative DNA damages, the guanine being oxidatived to 7,8-dihydro-8-oxogunine (8-OHdG) is most popular. 8-OHdG can exist stably and is easy to be detected, and it is concerned as biomarker of oxidative DNA damage. Besides, the most serious adverse effect of 8-OHdG on DNA is to induce G:C transversion to T:A because of the changes of DNA configuration. This transversion occurs early in carcinogenesis and is detected in oncogen ras and antioncogen p53 frequently. So the transversion is considered as closely related to carcinogenesis, aging of cells and some degeneration diseases.In human cells, the enzyme that can excise and repair 8-OHdG specifically is called 8-oxogunine DNA glycosylase (hOGG1). It can excise 8-OHdG opposited with C, and restore the normal G:C match in genome. hOGG1, which was cloned in 1997, plays an important role in keeping the stability of genome and preventing carcinogenesis.The study of epidemiology shows that hOGG1 has genetic polymorphology among different population, and is closely related to susceptibility of carcinogenesis. The mutation or loss of heterozygosity (LOH) of hOGG1 can increase the risk of tumor. So far, few studies on the relationship between hOGG1 and environmental carcinogens involved in limited kinds of materials are reported and some results are contrary. A great number of studies in this field are abroad while domestic studies are few. In this research, we want to construct cell strains with down-regulated expressed hOGG1 genes, which target to oxidative DNA damage and repair by ribozyme technology and molecular cloning technology. This will become a good toxicological tool for further study on the relationship between oxidative DNA damage and repair and tumor.Ribozyme technology is one of antisense nucleic acid technologies and a brand-new molecular biotechnology following gene-cloning skill. Ribozyme is a kind of RNA, which has endonuclease activity and can incise special mRNA sequence. A ribozyme designed reasonably can combine specifically with target mRNA, inhibit or block the expression of target genes through various mechanisms, and then have a biological effect on its target cells. Due to its particular merits, the studies about ribozyme, especially in the field of gene therapy, have been developing rapidly in the recent 10 years. But few applications of ribozyme in toxicology are reported.The research is divided into 4 parts, main contents are as follows:Part 1: The construction and identification of eukaryotic expression vector of hammerhead ribozyme targeting to hOGG1 mRNA. Computer was used to simulate the secondary structure of hOGG1 mRNA and to design ribozyme. Ribozyme gene was cloned into the eukaryotic expression vector pcDNA3.1(+), the eukaryotic expression vector of hammerhead ribozyme targeting to hOGG1 mRNA was constructed and identified by agarose gelelectrophoresis and DNA sequencing.Part 2: The construction and identification of cell strains with down-regulated expressed hOGG1 genes. The eukaryotic expression vector pcDNA3.1(+)-RZ was transfected into human A549 lung carcinoma cells through the transfection agent FuGENE 6, the transfected cells were screened by tolerance of hygromycin (G418), Reverse transcriptase-polymerase chain reaction (RT-PCR) targeting to Neo gene was used to identify the fulfill of transfection. In order to know the effect of inhibition of ribozyme to target gene, levels of hOGG1 mRNA were assessed relative to hGAPDH albeit without quantification relative to an internal standard.Part 3: The identification of biological characteristics of cell strains with down-regulated expressed hOGG1 genes. To learn the biological characteristics of cell strains with down-regulated expressed hOGG1 genes, the cell morphology, proliferation doubling-time, fraction distribution of cells in cell cycle, the basic apoptotic rate, the cell proliferation index, the number of clone formation in soft agar were also investigated, and the SOD activity was detected at the same time. The purpose of these studies was to provide the base of application.Part 4: The primary application in DNA oxidative damage and repair, sensitivity to anticancer medicines and 60Co-gamma rays. The differences of DNA damage and repair induced by Potassium dichromate (K2Cr2O7), hydrogen peroxide (H2O2), Adriamycin (ADM) and 60Co-gamma rays between A549 cells and A549-R cells were detected by single cell gel electrophoresis (comet assay). The cell viability after exposed to ADM and 60Co-gamma rays was detected by MTT assay. The fraction distribution of cells, the apoptotic rate and the cell proliferation index were detected by flow cytometry. The aim was to learn about the effect of the low expression of hOGGl on the sensitivity to anticancer medicines and 60Co-gamma rays.The Main results are as follows:1. Computer was used to simulate the secondary structure of hOGG1 mRNA and to design ribozyme. Ribozyme gene was cloned into the eukaryotic expression vector pcDNA3.1(+) by molecular cloning technology and was identified by agarose gel electrophoresis and DNA sequencing. The eukaryotic expression vector of hammerhead ribozyme targeting hOGGl was constructed successfully.2. The eukaryotic expression vector pcDNA3.1(+)-RZ was transfected into human A549 lung carcinoma cells through the transfection agent FuGENE6, the positive transfected cells were screened out by tolerance of G418. The evidence of successful transfection in these cells was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR) targeting to Neo gene. The expression of hOGGl mRNA in the positive transfected cells decreased 61.5% compared with the cells without transfection by RT-PCR. The results demonstrated that ribozyme gene could be transcripted into ribozyme RNA in cells and inhibit the expression of target gene hOGGl efficiently.3. At the early time of transfection, the cell morphology in transfected cells changed a bit. But the changes were not related with hOGG1. The morphology of the two kinds of cells tended to the same with the extension of the time. The growth characteristics including proliferation doubling-time in the two kinds of cells had no significant difference. The fraction distribution in cell cycle, the basic apoptotic rate, the cell proliferation index of the two kinds of cells had no significant difference, too. The number of clone formation in soft agar in transfected cells was less significantly than cells without transfection. However, the SOD activity in transfected cells was stronger than cells without transfection.4. The two kinds of cells had a bigger differences in DNA repair than in DNA damage. The two kinds of cells showed the same sensitivity to K2Cr2O7,H2O2, ADM and 60Co in the aspect of DNA damage excluding some exceptional dose or index. But the transfected cells showed lower repair capability to DNA damage induced by K2Cr2O7 and H2O2 than the cells without transfection. Both ADM and 60Co-gamma rays could induce DNA damage in two kinds of cells with good dose-response relationship. The transfected cells repaired DNA damage induced by ADM and Co-gamma rays more slowly than the cells without transfection. The results of flow cytometry displayed that both ADM and 60Co-gamma rays could induce GO/G1 arrest, increase of apoptotic rate, decrease of the cell proliferation index. The results of MTT test showed that the cell viability of the transfected cells was lower than that of the cells without transfection. All the results suggested that the transfection of ribozme targeting to hOGGl could increase the sensibility of cells to ADM and 60Co-gamma rays.