Relationship Between Polymorphism Of HOGG1 Gene And Coronary Artery Lesions In DM Patients And Effect Of Gene Overexpression In MtDNA On Repairing Oxidative Damage Induced By High Glucose In VECs

Part I Relationship between Polymorphism of h OGG1 Gene and Coronary Artery Lesions in DM Patients Objective:To study the relationship between h OGG1 Ser326 Cys gene polymorphism and severity of coronary artery lesions in patients with diabetes mellitμs. Methods:323 patients with diabetic mellitus underwent coronary angiography were retrospectively analyzed. These patients were carried out PCR-RFLP test, and were divided into Cys/Cys genotype(n=85), Ser/Ser genotype(n=117), and Ser/Cys genotype(n=121) according to the results of PCR-RFLP. All clinical data inclμding history of diseases, complicati- ons and biochemical markers, for example blood glμcose, blood lipids and so on were recorded. h OGG1 m RNA and 8-OHd G were mearsμred by RT-PCR and ELISA respectively. The results of coronary angiography were analyzed by two cardiovascμlar physicians, vessels nμmber and severity of coronary artery with lesions, Gensini score and SYNTAX score were detected by the unitary criteria. Results:1. Compared with Ser/Ser genotype and Ser/Cys genotype, 8-OHd G was higher but expression of h OGG1 m RNA was lower in Cys/ Cys genotype, and the difference was statistically significant(p<0.05). however, the difference of 8-OHd G and h OGG1 m RNA between Ser/Ser genotype and Ser/Cys genotype was not statistically significant(p>0.05).2. Probability of triple vessel lesions in Cys/Cys genotype was highest and probability of single vessel lesions in Ser/Cys genotype was lowest, but the difference of number of vessels with lesions among three genoty- pes was not statistically significant(p>0.05).3. Gensini score and SYNTAX score, the ratio of complex lesions in Cys/Cys genotype was all high er than the other two genotypes, and the difference was statistically significant(p<0.05); but the difference between Ser/Ser genotype and Ser/ Cys genotype was not statistically significant(p>0.05). Conclusions:h OGG1 Ser326 Cys gene polymorphism influences obvious sly the severity of coronary artery lesions in patients with diabetes mellitus,and coronary artery lesions in patients with diabetes mellitus carried Cys/Cys genotype are more severe. Its mechanism may be that the expression of h OGG1 m RNA in patients with diabetes mellitus carried Cys/Cys genotype is lower than the other two genotypes, then decrease the ability of identification and removal of 8-OHd G and the capacity of repairing DNA oxidative damage, and accelerate the development of atherosclerosis.Part II Effect of Oxidative Stress Damage Induced by High Glucose in Vascular Endothelial Cells in Vitro Environment Objective:To study oxidative stress damage induced by high glucose in vascular endothelial cells in vitro environment. Medoths:HUVECs were cultivated in DEMN culture medium containing 30mmol/L Dglucose, and the morphological changes of HUVECs were respectively observed with inverted fluorescence microscope before culture in DMEM culture medium with 30 mmol / L D-glucose and at 12 h, 24 h, 48 h after culture in DMEM culture medium with 30 mmol / L D-glucose, and at the same time the expression levels of SOD, h OGG1 m RNA and 8-OHd G in HUVECs at above time points were also tested, and damage degree of DNA and mt DNA in HUVECs were assessed Results:1. Along with the time of culture in DMEM with 30 mmol/L D-glucose, the morphological of HUVECs gradually turned sparse and less clear. And the cytoplasm and nucleus of cells varied in cellar morphology and the staining became weaker.2. In high glucose condition, the levels of 8-OHd G, ROS in HUVECs gradually increased with time prolonging, the oxidative stress damage of DNA and mt DNA induced by high glucose in HUVECs also increased with time of culture in in high glucose condition.3. And cellular DNA repair process for oxidative stress damage can also be enhanced, and the character was represented in the levels increase of h OGG1 m RNA expression. Conclusions:In high glucose condition, 8-OHd G, ROS in HUVECs gradually increased with time prolonging, and high glucose can also stimulate cellular repair process for oxidative injury. And all these resulted into changes of cellar morphology and function.Part III Effect of h OGG1 Gene Overexpression in Endothelial cells on Repairing Oxidative Damage Induced by High Glucose Section 1 Construction and Identification of HUVECs with Overexpression of hOGG1 Gene with Mitochondrial Transport Sequences Transfected by Adenovirus Objective:To construct HUVECs with overexpression of h OGG1 gene with mitocho- ndrial transport sequences for further researches by molecular biology technology. Methods:All experiments included the following 4 steps:1. MTS-h OGG1 gene was amplified by RT-PCR. And MTS-h OGG1 DNA fragments contain the mitochondrial transport sequences.2. Connection of MTS-h OGG1 gene and plasmid p ENTRTM3 C. MTS-h OGG1 Gene and plasmid p ENTRTM3 C was setted up restriction endonuclease reaction, and reco- mbinant plasmid p ENTRTM3C-MTS-h OGG1 were established correctly and the rec- ombinant plasmid was identified by restriction endonuclease analysis and RT-PCR amplification;3. Homologous recombination of recombinant plasmid and adenovirus vector. Take the recombinant plasmid p ENTRTM3C-MTS-h OGG1 and adenovirus vector p Ad/C- MV/V5-DEST for homologous recombination, positive clones were screened and identified by PCR, and the MTS-h OGG1 were established correctly.4. HUVECs were transfected with p Ad/CMV/V5-DEST-MTS-h OGG1 and p Ad/ CMV/V5-GW/lac Z, and HUVECS without transfection were used as control. After cultured for 48 hours, h OGG1 expression of three groups of cells were analyzed by using western blot technique. Results:1. Products of RT-PCR amplification with pc DNA3.1(+)-MTS-h OGG1 plasmid as a template are consistent with the purpose DNA fragments by agarose gel electrophoresis, and the products lenghth of electrophoretic bands is 1200 bp.2. p ENTRTM3C-MTS-h OGG1 were recombined with pc DNA3.1(+)-MTS-h O GG1 plasmid and p ENTRTM3 C plasmid. And requences of the testted gene were consistant with target gene by genetic screening. Agarose gel electrophoresis also displayed two electrophoretic bands of 2268 bp and 1200 bp after double digestion with Bam H I and Xho I, and the bands of 2268 bp was the linear fragments of the original plasmid of p ENTR3 C, and the bands of 1200 bp were the inserted gene fragments. Recombinant plasmid was successfully constructed.3. h OGG1/β-actin in HUVECs with p Ad/CMV/V5-DEST-MTS-h OGG1(target gene group), p Ad/CMV/V5-GW/lac Z(blank gene group) and HUVECS without any transfection(blank control group) was tested and was respectively 1.02±0.12, 0.51±0.08,0.53±0.07, and the expression of h OGG1/β-actin had statistically significant differences in above 3 groups. Conclusions:Overexpression of h OGG1 gene with mitochondrial transport sequences in HUVECs can be successfully constructed by molecular biology techniques using adenovirus vectors p Ad/CMV/V5-DEST Section 2 The Study of overexpression of mitochondrial h OGG1 gene in VECs on repairing oxidative stress damage induced by high glucose ObjectiveTo study the effect of overexpression of mitochondrial h OGG1 gene on repairing oxidative stress injury induced by high glucose in HUVECs. Method:HUVECs transfected with p Ad/CMV/V5-DEST-MTS-h OGG1(target gene group), HUVECs transfected with p Ad/ CMV/V5-GW/lac Z(blank gene group) and HUVECs without any transfection(blank control group) were cultured respectively in DMEM culture medium with 30 mmol /L D-glucose, ROS and 8-OHd G was detected before culture in DMEM culture medium with 30 mmol/L D-glucose and at 12 h, 24 h, 48 h after culture in DMEM culture medium with 30 mmol/L D-glucose. ROS, 8-OHd G and change of mitochondrial membrane potential in HUVECs was respectively evaluated, the oxidative stress damage of HUVECs DNA induced by high glucose in HUVECs were also evaluated in this study. Results:1. With the culture time prolonging, ROS, 8-OHd G in three groups of HUVECS cultured in high glucose medium with 30 mmol/L glucose had no significant difference at the same time(P >0.05). But ROS, 8-OHd G in target gene group were less than the other groups(P<0.05), and ROS, 8-OHd G in blank gene group and blank control group had no singnificant difference(p>0.05).2. After cultured in high glucose medium with 30mmol/ L D-glucose for 12 h, 24 h and 48 h respectively, ratio of average fluorescence intensity representing the change of mitochondrial membrane potential in target gene group were higher than the other two groups(P<0.05); but the ratio in blank gene group and blank control group had no significant difference(p>0.05). Conclusions:Overexpression of mitochondrial h OGG1 gene in HUVECs plays a role in the repair of oxidative stress injury induced by high glucose, and can attenuate oxidative stress injury...