Objective To observe the oxidative damage of mtDNA, apoptosis and expression of adhesion molecules in retinal capillary cells of diabetic rat with different disease courses. Methods One hundred Sprague-Dawley rats were randomly divided into the control group and the experimental group. The rats of experimental group were induced with streptozotocin (STZ) injection creating a diabetic model. Then they were divided into DR1m, DR2m DR3m group according to disease courses. The rats of control group were divided into NR1m, NR2m, NR3m group. Rat retinal capillaries were prepared, and then the contents of undamaged mtDNA were examined by Southern blot combined with Fpg. The expression of cyclooxygenase (COX)-1 encoded by mtDNA and transcription factors A (mtTFA) mRNA were detected by real-time quantitative polymerase chain reaction (RT-PCR). Apoptosis and expression of intercellular adhesion molecule-1 (ICAM-1) were detected by terminal dUT nick endlabeling (TUNEL) immuno-fluorescence and immunohistochemistry respectively. Results The contents of undamaged mtDNA in rats of DR1m, DR2m, DR3m were less than those of NR1m、NR2m、NR3m. The contents of undamaged mtDNA in diabetic rats decreased with the increase of disease courses. In addition, the mRNA levels of COX-1 and mtTFA were downregulated in diabetic rats. The positive cells of TUNEL and ICAM-1TUNEL and ICAM-1 in diabetic rats increased with the increase of disease courses. Conclusion With the increase of disease courses, mtDNA damage and apoptotic cells are increased, while the expression of mRNA encoded by mtDNA and ICAM-1 decreased in retinal capillary cells in diabetic rats.
Objective To construct specifically expressed vascular endothelial growth factor (VEGF)165 gene in retina. Methods Rho promoter, specifically expressed in retina, was amplified by polymerase chain reaction (PCR) from the genomic DNA of a BLAB/C rat, then it was cut with restriction enzymes and cloned into the plasmid pcDNA3.1+-VEGF165 to form recombinant plasmid pcDNA3.1+-rho-VEGF165. The correct recombinant plasmid pcDNA3.1+-rho-VEGF165 was identified by restriction enzymes and PCR, and was transferred by jetPEI into cultured human navel vein endothelial cells and human retinal pigment epithelial (RPE) cells. The expression of VEGF protein in human navel vein endothelial and RPE cells was detected by immunocytochemical staining and protraction of the growth curve of the cells. Results In human RPE cells, the expression of VEGF protein was more in recombinant plasmidpcDNA3.1+-rho-VEGF165 than that in plasmidpcDNA3.1+-rho-VEGF165 ; in human navel vein endothelial cells, no obvious difference of the expression of VEGF protein between recombinant plasmid pcDNA3.1+-rho-VEGF165 and plasmid pcDNA3.1+-rho-VEGF165 was found. Conclusions The construction of pcDNA3.1+-rho-VEGF165 carrier may provide the basic material for the study of the nosogenesis of VEGF in retinal neovascularization, and establish the foundation to set up the model of transgenic mice with VEGF specific expressing in retina. (Chin J Ocul Fundus Dis, 2005,21:106-108)
ObjectiveTo investigate the difference of DNA methylation before and after bariatric surgery.MethodThe relevant literatures of the research on the changes of DNA methylation level and gene expression regulation in blood and tissues before and after bariatric surgery were retrieved and reviewed.ResultsDNA methylation was an important method of epigenetic regulation in organisms and its role in bariatric surgery had been paid more and more attention in recent years. Existing studies had found that there were changes of DNA methylation in blood and tissues before and after bariatric surgery. The degree of methylation varies with different follow-up time after bariatric surgery and the same gene had different degrees of methylation in different tissues, and some even had the opposite results.ConclusionsDNA methylation levels before and after bariatric surgery are different in different tissues. And studies with larger sample size and longer follow-up time are needed, to further reveal relationship among DNA methylation, obesity, and bariatric surgery.
Objective To investigate the effects of QUE on proliferation and DNA synthesis of cultured retinal pigment epithelium(RPE) cells with or without EGF. Methods With or without EGF, cultured RPE cells were treated with QUE by various concentrations(200,100,50,1mu;mol/L) and with QUE 200mu;mol/L at different times(24-168 hr), cells proliferation and DNA synthesis were evaluated by cell count method and the uptake of thymidine. The viability of cells was determined by trypanblue exclusion. Results The best concentration of QUE which inhibits proliferation and DNA synthesis of PRE cells was 200mu;mol/L. The significant inhibition effect of QUE occurred at 48hr, and the best inhibition of QUE occurred at 96hr. QUE had more powerful effect of antiproliferation on RPE cells, and the viability of RPE cells was over85%. Conclusion The results suggested that QUE could inhibit the proliferation of RPE cells in a dose-dependent and time-dependent manner, especially inhibit the proliferation induced by EGF stimulating. QUE had no cyto-toxic effect on RPE cells cultured in vitro. (Chin J Ocul Fundus Dis,1999,15:27-29)
Microorganism distributes in the organs of human body which connect with external environment, especially those organs in the gastrointestinal tracts, and it also plays a fundamental role in the physiopathology of the host's body. Because the microorganism is very small and has a great variety, it is difficult to reveal the significance of microorganism in the human physiopathology comprehensively and deeply. With the development of molecular biology, genomics, bioinformatics and other disciplines, the microbiome research will be more possible and easier. There are two key contents of microecology. One of these is to identify and quantify the diversity of microorganism, and the other is to reveal activity and the physiopathological function of microorganism in the host. Microbiome research methods, therefore, can be summarized as the traditional detection methods, construction of gene library, the genetic fingerprint analysis and molecular hybridization techniques and so on.
ObjectiveTo detect the inhibitory effect of early growth response gene-1 DNA enzyme (EDRz) on proliferation of vascular smooth muscle cell (VSMC) and intimal hyperplasia, and confirm the effect of gene therapy on stenosis and occlusion after vein transplantation. MethodsEDRz was constructed, and autogenous vein graft model was established with Wistar rats, transplanting the right jugular vein to infra renal abdominal aorta by microsurgical technique. EDRz was transfected to the graft veins and the vein graft samples were harvested at hour 1, 2, 6, 24 and on day 3, 7, 14, 28, 42 after grafting, 10 Wistar rats were randomly selected in every time. The expression of EDRz in transfected vein graft was detected by fluorescent microscope. Egr-1 mRNA was measured by reverse transcription-PCR (RT-PCR) and in situ hybridization, respectively. The protein expression of Egr-1 was detected by Western blot and immunohistochemistry, respectively. HE stained vein grafts were observed under microscope. Results① The results of EDRz transfected vein graft: At hour 1 after grafting, EDRz was mainly located in adventitia, tunica media, and partial endothelial cells of vein graft; At hour 2, 6, and 24, EDRz was located in tunica media of vein graft; and on day 7, it was mainly located in intima of vein graft. There wasn’t EDRz in vein grafts on day 14, 28, and 42. ② The results of expression of Egr-1 mRNA: Detection by RT-PCR: At hour 1 after transfecting, the expression of Egr-1 mRNA arrived at the peak, and declined at hour 2, 6, and 24. The expression was tenuity on day 3. Egr-1 mRNA expression was not found on day 7, 14, 28, and 42. The expression of Egr-1 mRNA at hour 1 was significantly higher than that of the other time point (Plt;0.01). The result of in situ hybridization was coincident with RT-PCR. ③ The results of expression of Egr-1 protein: The result of Western blot: There was no expression of Egr-1 protein in normal veins. At hour 2 after grafting, expression of Egr-1 protein was found, and declined at hour 6, 24, and on day 3. There was no expression of Egr-1 protein at hour 1, and on day 7, 14, 28, and 42. The expression of Egr-1 protein at hour 2 was significantly higher than that of the other time point (Plt;0.01). The result of immunohistochemistry was coincident with Western blot. ④The degree of VSMC hyperplasia and intimal thickness were lighter in EDRz transfected vein grafts than that in nottransfected vein grafts contemporarily. ConclusionsEDRz could reduce the expression of Egr-1 in autogenous vein graft, and could effectively restrain VSMC proliferation and intimal hyperplasia, and prevent vascular stenosis and occlusion after vein grafting.
PURPOSE:To investigate mitochondrial DNA(mtDNA) of Leber's hereditary optic neuropathy(LHON). METHODS:Polymerase chain reaction(PCR)method was used to analyse mtDNA of 11 patients in a pedigree with LHON and 4 control subjects from none LHON pedigree. RESULTS:There was a loss of a restriction site for the restriction endonuclease SfaN.Ⅰin Ihe Patients with LHON. In this pedigree,maternal lineage was regarded a carrier of the pathogenic gene. CONCLUSIONS:The patients with Leber's hereditary optic neuropathy have a point mutation in mtDNA,which results in loss ol SfaN I endonuclease restriction site .and this change is one of mechanisms inducing this disaese. （Chin J Ocul Fundus Dis,1997,13: 27-29）
Objective To evaluate branched-chain DNA (b-DNA) signal amplification and semi-quantitative (Sq) RT-PCR in detection of free cancer cells in peritoneal flushing fluid of colorectal cancer patients during surgery. Methods The CEA mRNA in peritoneal flushing fluid in 48 cases of colorectal cancer were detected by b-DNA and SqRT-PCR. Peritoneal flushing fluid cytology (PLC) was conformed simultaneously to detect the free cancer cells. The peritoneal flushing fluid of 12 cases with colorectal benign disease were taken as negative control, GAPDH mRNA as internal control. Results In colorectal cancer patients, positive rate of free cancer cells by bDNA and SqRT-PCR （43.8%, 31.3%） was higher than that by PLC （4.2%）. The relative quantitative expressions of CEA mRNA were related to the Dukes staging， depth invasion and differentiation degree (Plt;0.05)， but irrelevant to tumor size，the patients’ age and gender (Pgt;0.05).Conclusion Both b-DNA and SqRT-PCR technologies have advantages and disadvantages to detect free cancer cells in peritoneal flushing fluid， which are related to clinicopathological factors.
【Abstract】ObjectiveTo study the effect of transfection with antisense DNMT3b gene eukaryotic expression vector on the expression of DNMT3b gene in human cholangiocarcinoma cell line QBC-939. MethodsThe constructed antisense DNMT3b gene eukaryotic expression vector was transfected into the human cholangiocarcinoma cell line QBC-939 by using lipofectamine transfection reagents, and positive cell clones were obtained by using G418 selection after transfection. Whether the constructed recombinant vector was transfected into QBC-939 cells successfully was confirmed by amplifying the exogenous neoR gene with PCR method. The expression of DNMT3b gene mRNA and protein were detected by semi-quantitative RT-PCR and FCM methods respectively. ResultsFollowing the transfection of antisense DNMT3b gene eukaryotic expression vector, the mRNA level of DNMT3b gene in QBC-939 cells of human cholangiocarcinoma decreased from 0.956±0.053 to 0.209±0.023, and the protein level of DNMT3b gene also decreased from (75.38±3.22)% to (29.87±3.46)%. There were very significant differences on the expression levels of DNMT3b gene between non-tranfections group and the antisense DNMT3b gene eukaryotic expression vector transfection group (P＜0.01). ConclusionTransfection with antisense DNMT3b gene eukaryotic expression vector significantly reduces the expression level of DNMT3b gene in human cholangiocarcinoma cell line QBC-939, and this study may provide a valid tool and method to investigate the function of DNMT3b gene and its role in cholangiocarcinoma.
Objective To analyze the relationship between genotype and phenotype of vitelline macular dystrophia (VMD2) gene in a family with Best disease, and to provide the theoretical basis for gene diagnosis of Best disease. Methods Mutation in the coding regions and the promotor sequence of VMD2 gene from 10 members in a family with Best disease were screened by polymerase chain reaction (PCR) and direct DNA sequencing, and combined with a conformation sensitive gel electrophoresis (CSGE) approach, VMD2 gene screening was performed on 100 normal control individuals. Results In the 10 members, Trarr;C nucleotide change at the 223 base of exon 3 was detected in 9, including 6 with Best disease who was confirmed by ophthalmoscopy and electrophysiological examination in whom 2 were affirmed as having homozygote of this mutation. Other 3 young family members with VMD2 gene mutation only had abnormal electro-oculogram manifestations. Above mutation was not detected in the normal control individuals. Conclusions The phenotype and genotype of VMD2 in the family with Best disease is highly correlated. Mutation in VMD2 gene is the nosogenesis in this family. Mutation screening of VMD2 gene can be used for genic diagnosis and genetic consultation of Best disease. (Chin J Ocul Fundus Dis, 2006, 22: 86-89)