过氧化氢酶基因重组腺病毒对大鼠晶状体氧化损伤的防护作用

The protective effect of recombinant adenovirus of catalase on oxidative damaged rat lens

目的探讨过氧化氢酶(CAT)基因重组腺病毒对晶状体氧化损伤进行基因治疗的可行性。方法克隆CAT基因,构建CAT基因重组腺病毒,测定病毒液滴度。体外培养大鼠晶状体,Western印迹分析确定重组腺病毒感染大鼠晶状体后CAT表达峰值时间。按随机分组原则将大鼠晶状体分为对照组(A组)、H2O2处理组(B组)、H2O2及重组腺病毒处理组(C组),分别于6、12、18、24h检测晶状体透明度,并采用双染色流式细胞技术进行细胞凋亡分析。结果构建出具有生物学活性的CAT基因重组腺病毒。CAT基因重组腺病毒感染体外培养大鼠晶状体后9h,CAT表达达到峰值,并在36h内保持恒定。6、12、18、24h,C组晶状体的透明度均低于A组,高于B组,3组比较差异均有统计学意义(P<005);C组细胞凋亡率均高于A组,低于B组,3组比较差异均有统计学意义(P<005)。结论CAT基因重组腺病毒可抑制氧化剂导致的晶状体混浊和晶状体上皮细胞凋亡,具有用于治疗氧化所致白内障的可能性。

Objectives To investigate the validity of catalase recombinant adenovirus on the treatment of oxidative cataract. Methods The coding sequence of catalase was cloned and the catalase reombinant adenovirus was constructed. The expression time course of catalase gene in rat lens infected by recombinant adenovirus was determined by Western blotting. Cultured rat lens were randomly 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. The transparence and apoptosis ratio of lens on the time points of 6, 12, 18,24 hours were determined by image analysis and double colour flowcytometry. Results The coding sequence of catalase was cloned and recombinant adenovirus was successfully constructed. The expression of catalase in cultured rat lens infected by recombinant adenovirus reached peak point on 9 hours post infection and maintained the level in the whole experiment period. The transparence of the lens in the group treated by hydrogen peroxide combined with catalase recombinant adenovirus was higher than that of group treated by hydrogen peroxide and lower than that of the control group on the time points of 6, 12, 18,24 hours post infection. The differences among groups were statistically significant (P<0.05). On the same time points, the apoptosis ratio of the group treated by hydrogen peroxide combined with catalase recombinant adenovirus was lower than that of the group treated by hydrogen peroxide and higher than the control group. The differences among groups were statistically significant (P<0.05). Conclusion^The catalase recombinant adenovirus, which can inhibit the turbidity and cell apoptosis of lens caused by oxidant, may be used as the gene therapy of oxidative cataract.(Chin J Ophthalmol,2005,41:~156-160)