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The Study on the Relationship between Caveolin-1 and SelS Protecting ECV304 from the Injuring by H2O2

Author: ZhaoYin
Tutor: DuJianLing
School: Dalian Medical University
Course: Internal Medicine
Keywords: Endothelial dysfunction SelS / Tanis gene pcDNA3.1-SelS Human umbilical vein endothelial cell line Caveolin -1
CLC: R362
Type: Master's thesis
Year: 2008
Downloads: 50
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Abstract


The purpose: gene fragment was cloned from human adipose tissue SelS, build the pcDNA3.1-SelS eukaryotic expression carrier. The the pcDNA3.1-SelS Instantly transfection in human umbilical vein endothelial cells (ECV304), respectively, from the mRNA and protein levels of detection SelS gene expression in ECV304 cells. Transient transfection will not transfected ECV304 cells were divided into group the, SelS transfection group the (SelS high expression group) and the empty vector transfected group before and after the injury to different concentrations of hydrogen peroxide (H2O2), observed in each group cell damage oxidative stress indicators and new experimental evidence for differences in levels of expression of caveolin -1 (Caveolin-1, Cav-1) to investigate whether Caveolin-1 anti-oxidation and SelS depth the study SelS endothelial protection mechanism . Method: 1. Using RT-PCR from human adipose tissue to get SelS gene fragment (79 to 645bp) was cloned in the pcDNA3.1 eukaryotic expression vector through two subclones eukaryotic expression vector plasmid pcDNA3 the .1-SelS, parallel EcoRI / XhoI restriction enzyme digestion and sequencing. Application of liposomal transfection technology of pcDNA3.1-SelS and pcDNA3.1 transient transfection of ECV304 cells to ECV304 cells divided into SelS transfected group and empty vector transfected group, at the same time to untransfected group ECV304 cells as a control. GAPDH as the internal reference to the semi-quantitative detection SelS mRNA level of expression; β-actin as an internal reference, the Western blot was used to detect SelS protein levels of expression. 3 cells in each group were treated with final concentration of 0,400,600,800,1000 μmol / L H2O2 in culture medium for 6 hours after four methyl thiazolyl tetrazolium (MTT) observed the ability of H2O2 on cell proliferation impact; different concentration of cell supernatants thiobarbituric acid method for the determination of the content of malondialdehyde (MDA), superoxide dismutase (SOD) activity was measured by xanthine oxidase. 4 according to the MTT results, select a moderate reduction of cell survival containing H2O2 concentration of 800μmol / L of culture medium cells 6 hours after the extraction of each group of total cellular RNA by RT-PCR method to detect the expression of Caveolin-1 mRNA. Results: 1. Total RNA extracted from human adipose tissue is complete, Sels gene cDNA sequencing results from Genebank sequence (NM-018445) consistent. XhoI and EcoRI restriction analysis and sequencing confirmed the pcDNA3.1-SelS eukaryotic expression vector plasmid was successfully constructed. 2.RT-PCR, Western blot testing confirmed SelS endogenous expression in ECV304 cells, while the semi-quantitative analysis shows SelS the transfection group SelSmRNA and protein expression levels compared with untransfected and empty vector transfected group was significantly liter high (P lt; 0.01), untransfected and empty vector transfected group showed no differences (P lt; 0.01). Cell survival rate (%): ECV304 cells exposed to 6 hours in a culture medium containing different concentrations of H2O2, cell survival rate was significantly decreased; concentration of H2O2 800,1000 μmol / L, SelS transfected group (34 ± 10,27 ± 15) cell survival rate was significantly higher than the non-transfected group (32 ± 12,24 ± 9) and empty vector transfected group (31 ± 14,23 ± 10) (P lt; 0.01), empty vector transfection group compared with untransfected group difference was not significant (P gt; 0.05). 4.MDA content (nmol / mL): each group of cells in the supernatant of MDA content increased with the increased concentration of H2O2; concentration of H2O2 600,800,1000 μmol / L, SelS transfected group MDA levels (3.50 ± 0.07,6.30 ± 0.07,10.50 ± 0.08) were lower than the non-transfected group (6.40 ± 0.03,10.40 ± 0.11,18.00 ± 0.07) and the empty vector transfected group (5.80 ± 0.08,9.00 ± 0.09, 16.40 ± 0.19), H2O2 concentration difference 1000μmol / L more significantly (P lt; 0.05, P lt; 0.05, P lt; 0.01), empty vector transfected group and untransfected group compared differences not statistically significant (P gt; 0.05). The SOD vitality in the 5.SOD vitality (U / ml): each group of cells in the supernatant with the higher concentration of H2O2 weakened; 600,800,1000 μmol / L H2O2 concentration, SelS SOD activity in transfected group (8.34 ± 0.16,7.48 ± 0.87,6.60 ± 1.58) was significantly higher than the non-transfected group (7.52 ± 0.29, 5.70 ± 0.22, 4.62 ± 2.33) and empty vector transfected group (7.12 ± 1.63,6.10 ± 1.78,5.00 ± 1.27), H2O2 concentration 800,1000 μmol / L increased more significantly (P lt; 0.05, P lt; 0.01, P lt; 0.01); empty vector transfected cells compared with untransfected group difference (P gt; 0.05). 6.Caveolin-1 mRNA expression: ECV304 cells were exposed to six hours in a culture medium containing different concentrations of H2O2, compared with before the injury, not transfected group (0.74 ± 0.01 vs 0.48 ± 0.01, P lt; 0.01) the SelS transfection group (0.67 ± 0.01 vs 0.63 ± 0.01, P lt; 0.05) and empty vector transfected group (7.12 ± 1.63 vs 0.58 ± 0.01, P lt; 0.01) was significantly higher; H2O2 injury, SelS transfected group (0.67 ± 0.01) was significantly lower than the non-transfected group (0.74 ± 0.01) (P lt; 0.01) and empty vector transfected group (7.12 ± 1.63) (P lt; 0.01), but not to turn the the stained group with empty vector transfection group was no significant difference (P gt; 0.05). Conclusion: 1. Successfully constructed the eukaryotic expression vector pcDNA3.1-SelS will be the pcDNA3.1-SelS transiently transfected into ECV304 cells, and confirmed in SelS the endogenous expression of the ECV304 cells. High expression SelS can be significantly reduced H2O2 inhibition of endothelial cell growth, to reduce the generation of oxidative stress MDA, enhanced activity of SOD has a protective effect on endothelial cells. 3. H2O2 raised ECV304 cells Caveolin-1 expression, the high SelS expression can inhibit Caveolin-1 upregulation, Description SelS protection ECV304 cells from H2O2 injury Caveolin-1.

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