| ObjectiveMechanism of age related cataract was complex and closely related with oxidative stress. Tthioltransferase (TTase) played a key role in the body's antioxidant defense system. The expression of TTase in lens epithelial cells affected lens the balance state of redox and the stability of protein conformation,and was closely related to the maintenance of lens transparency.This experiment study the expression and activity of TTase in lens epithelial cells of cataract and normal human in the molecular level and protein level, to explore the mechanism of cataract and for the further study of drug prevention and treatment of cataract to provide a new clue.MethodsThe anterior capsule membrane from phacoemulsification and continuous curvilinear capsulorhexis in age related cataract patients was collected as the experimental groups,110 eyes of 104 cases,and divided into the three groups by the clinical types of cataract including cortical cataract, nuclear cataract, and subcapsule cataract. Cortical cataract were 38 eyes of 35 cases, nuclear cataract 42 eyes of 39 cases, subcapsule cataract 30 eyes of 30 cases, samples preserved in liquid nitrogen immediately. The anterior capsule membrane in transparent lens was collected as control group by the same way, 12 eyes of 9 cases. Inclusion criteria: the experimental groups from age-related cataract patients diagnosed and divided to sub-experimental groups by an experienced ophthalmologist, except for the history of high myopia, exposure to radiation for a long time, and other eye diseases and systemic diseases; the control group from the ocular trauma patients with transparent lens dislocation, or the body excluded other eye diseases.Each group's TTase mRNA and protein expression was detected with Western-blotting and RT-PCR technology. The difference expression of TTase among the groups were compared withβ-actin as the reference.The data was expressed as mean±standard deviation ( x±s), analyzed by SPSS10.0 statistical software, p < 0.05 as statistically significant difference.Results1,Western-blotting analysis: SDS-PAGE and Western-blotting analysis showed molecular weight of TTase was about 11KDa;lens epithelial cells under the lens anterior capsule membrane of experimental groups and control group expressed TTase protein in varying degrees;2,PCR amplification: Lens epithelial cells under the lens anterior capsule membrane of experimental groups and control group were detected in the expression of TTase andβ-actin, which the length of gene amplification products was correspond to nucleic acid sequences in GenBank;3,RT-PCR: Ratios of TTase/β-actin mRNA of lens epithelial cells under the lens anterior capsule membrane were cortical cataract group 1.36±0.16, nuclear cataract group 1.03±0.11, subcapsule cataract group 1.46±0.12, and control group 2.01±0.14. The statistically analysis results showed that expression of TTase mRNA in lens epithelial cells under the anterior capsule membrane of experimental groups was decreased significantly in comparison with control group ( p <0.05).There was a significant difference between cortical and nuclear cataract, nuclear and subcapsular cataract, respectively ( p <0.05).There was no significant difference between cortical cataract and subcapsular cataract ( p > 0.05).Conclusion1,TTase were expressed in lens epithelial cells under the lens anterior capsule membrane of age related cataract groups and lower than normal control group;2,The expression of TTase was different among cortical cataract, nuclear cataract, and subcapsule cataract,lower nuclear expression;3,In lens epithelial cells under the lens anterior capsule membrane of age related cataract TTase activity wasn't linear relationship with age. |
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