| Objectives1. To investigate the mechanism of lens epithelial cells apoptosis induced by reactive oxidant species.2. To investigate the variant of the toleration to oxidative damage of lens epithelial cells stably transfected with catalase gene.3. To investigate the validity of catalase recombinant adenovirus on the therapy of oxidative cataract.Methods1. The human lens epithelial cells SRA01/04 were cultured in DMEM medium with 15% FBS, 37t ,5% C02.2. The coding sequence of catalase was cloned and verified by DNA sequencing.3. The expression vector of catalase, named pIRESneo3 vector, was constructed and the inserted site of catalase in pIRneo3 vector was verified by double enzyme digested.4. The vector was introduced into SRA01/04 cells by transfaction.5. The durable concentration of SRA01/04 cells to G418 was detected and stable expression clones were screened and amplified by medium contained G418.6. The expression of catalase in the transfected SRA01/04 cells and un-transfected SRA01/04 cells was determined by Western blotting.7. Peroxide hydrogen was used to treat both kinds of cells. MTT method wasused to determine the IC5 of H202 to lens epithelial cell.8. FACS and AO/EB stain were used to analysis the apoptosis of lens epithelial cells induced by H202.9. The distribution of cytochrome C was detected by immunohistochemistry.10. The activation of caspase-3 was tested by Western blotting.11. The catalase reombinant adenovirus was constructed and the viral liter was determined.12. The rat lens were cultured in DMEM medium with 10% FBS, penicil-line (10 ~n U/L) , streptomycin (10 7f U/L) , 37癈 ,5% C02.13. The expression time course of catalase gene in lens infected by reombinant adenovirus was determined by Western blotting.14. Lens were divided into 3 groups; the control group, the group treated by hydrogen peroxide and the group treated by hydrogen peroxide combined with catalase recombinant adenovirus.15. The transparence and apoptosis ratio of lens on the time points of 6hrs, 12hrs,18hrs,24hrs were determined by image analysis and duble colour flowcy-tometry respectively.Results1. The CDS of catalase was cloned successfully and inserted into the right position in pIRESneo3 vector.2. A stable expression clone, named SRA01/04-CAT, was acquired. The expression level of catalase in SRA01/04-CAT cells was 3.65 times higher than that in SRA01/04 cells. The activity of catalase in SRA01/04-CAT cells was 2. 2 times higher than that in SRA01/04 cells.3. The IC5 of H202 to SRA01/04-CAT was 85.65ji,M and the IC5 of H-A to SRA01/04 was 32. 24u,M. The toleration to oxidative damage of SRA01/04-CAT cells was 2.65times higher than untransfected cells.4. During the apoptosis process of lens epithelial cells, cytochrome C was released from mitochondria to cytoplasm. Caspase-3 was activated in the process too. Comparing with SRA01/04, SRA01/04-CAT cells had a stronger resistanceto H202 because the releasing of cytochrome C was less and the activation of caspase-3 was weaker in them.5. The catalase gene recombinant adenovirus was successfully constructed and the viral liter was 4 x 1012pfu/ml.6. The expression of catalase in lens infected by recombinant adeno virus reached peak point on 9hrs post infection and maitained the level in the whole experiment period.7. On the same time point, the transparence of the group treated by peroxide hydrogen combined with catalase recombinant adenovirus was higher than that of group treated by peroxide hydrogen and lower than control group. Meanwhile, the apoptosis ratio of the group treated by peroxide hydrogen combined with catalase recombinant adenovirus was lower than that of group treated by peroxide hydrogen and higher than control group. The differences among groups were significant.Conclusion1. The mitochondria damage may properbly underly the apoptosis of lens epithelial cells induced by reactive oxidant species. Enhencing the reactive oxidant species eliminating capacity of lens epithelial cells can restrain...
|
PDF Full Text Request