Expression of superoxide dismutase in whole lens prevents cataract formation.
Expression of superoxide dismutase in whole lens prevents cataract formation.
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发表时间：
2005-10
期刊：
影响因子：
2.2
通讯作者：
Dingbo Lin;Micheal E. Barnett;L. Grauer;J. Robben;Annie Jewell;L. Takemoto;D. Takemoto
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文献类型：
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作者：
Dingbo Lin;Micheal E. Barnett;L. Grauer;J. Robben;Annie Jewell;L. Takemoto;D. Takemoto
氧化损伤是白内障的主要致病因素，白内障约占全世界人类致盲病例的一半。在这项研究中，我们希望确定是否超氧化物歧化酶（SOD）在完整的晶状体过表达可以防止白内障的形成氧化应激。方法将6周龄雄性/雌性Sprague道利大鼠的新鲜完整晶状体与编码人SOD 1（Cu/Zn-SOD）基因的质粒DNA在37 ℃、含95%空气和5%CO2的CO2细胞培养室中孵育。用Western blotting和SOD酶活性测定SOD 1的表达。用H2 O2处理有或没有SOD 1过表达的晶状体，并检查白内障形成。通过PKC γ酶活性测定来确定SOD 1对蛋白激酶C γ（PKC γ）的调节。完整的透镜间隙连接通过染料转移测定来确定。结果在透镜过表达系统中，SOD 1 cDNA与EYFP融合，产生EYFP：SOD 1融合蛋白，其允许从内源性SOD 1检测。用质粒DNA孵育完整晶状体产生EYFP：SOD 1融合蛋白，如使用抗GFP或抗SOD 1抗体通过蛋白质印迹测定的。这引起SOD酶活性的显著增加。数据表明，SOD 1质粒DNA可以在培养的完整晶状体中表达为功能酶。过表达SOD 1的晶状体在100 μ M的H2 O2处理24小时后保持透明，与对照相似。SOD 1的过表达减弱了H2 O2对PKC γ激活和随后的间隙连接抑制的作用，表明SOD 1的过表达可能减少活性氧（ROS）的产生，这将阻止H2 O2对白内障形成的正常作用。结论：在整个透镜中过表达SOD 1可防止H2 O2诱导的透镜氧化损伤（白内障形成），并防止随后由蛋白激酶C γ对间隙连接的控制。
PURPOSE Oxidative damage is a major factor causing cataracts, which account for almost half of human blindness cases worldwide. In this study, we wished to determine if overexpression of superoxide dismutase (SOD) in intact lenses could prevent cataract formation induced by oxidative stress. METHODS Fresh, intact lenses from 6-week-old male/female Sprague Dawley rats were incubated with plasmid DNA encoding the human SOD1 (Cu/Zn-SOD) gene at 37 degrees C in a CO2 cell culture chamber with 95% air and 5% CO2. SOD1 expression was determined by western blotting and SOD enzyme activity. Lenses with or without overexpression of SOD1 were treated with H2O2 and cataract formation was examined. SOD1 regulation of protein kinase Cgamma (PKCgamma) was determined by PKCgamma enzyme activity assay. Intact lens gap junctions were determined by dye transfer assay. RESULTS In the lens overexpression system, SOD1 cDNA was fused to EYFP to generate EYFP:SOD1 fusion proteins which allow detection from endogenous SOD1. Incubation of intact lenses with plasmid DNA produced EYFP:SOD1 fusion proteins as determined by western blot using anti-GFP or anti-SOD1 antibodies. This caused significant increases in SOD enzyme activity. Data indicated that SOD1 plasmid DNA can be expressed as a functional enzyme in intact lenses in culture. Lenses overexpressing SOD1 remained clear after H2O2 treatment at 100 muM for 24 h, similar to control. Overexpression of SOD1 diminished the effect of H2O2 on PKCgamma activation and subsequent inhibition of gap junctions, indicating that overexpression of SOD1 may reduce reactive oxygen species (ROS) production, and this would prevent the normal H2O2 effect on cataract formation. CONCLUSIONS Overexpression of SOD1 in whole lens prevents H2O2-induced oxidative damage (cataract formation) to the lens and subsequent control of gap junctions by protein kinase Cgamma.