【摘 要】
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目的 构建具有特异阻断大鼠NgR基因功能的siRNA表达系统,为视神经损伤基因治疗提供新的方法 .方法 实验研究.根据Genbank提供的NgR基因mRNA序列,应用设计软件设计特异性的短
【机 构】
：
第三军医大学大坪医院野战外科研究所眼科
论文部分内容阅读
目的 构建具有特异阻断大鼠NgR基因功能的siRNA表达系统,为视神经损伤基因治疗提供新的方法 .方法 实验研究.根据Genbank提供的NgR基因mRNA序列,应用设计软件设计特异性的短链寡核苷酸,化学合成2对编码短发夹RNA的寡核苷酸序列,退火、克隆到经BglⅡ、Hind Ⅲ酶切处理的pSUPER-EGFP质粒,获得重组siNgR质粒,用EcoR Ⅰ和HindⅢ双酶切和序列测定对重组体进行鉴定,最后将构建的表达载体转染Wistar大鼠体内视网膜神经节细胞,免疫印迹法观察对NgR蛋白表达的影响.结果 重组siNgR表达载体的酶切鉴定结果 和测序结果 表明重组载体构建成功.视网膜冰冻切片后,经荧光显微镜观察,视网膜神经节细胞层和视神经纤维可见绿色荧光蛋白表达.免疫印迹法分析表明,siNgR-1和siNgR-2可抑制RGCs NgR蛋白的表达,而siNgR-c和pSUPER-EGFP对照对NgR蛋白的表达无明显影响.结论 成功构建了siRNA表达载体,此载体具有阻断NgR基因的表达的功能,为进一步研究视神经损伤基因治疗打下基础.(中华眼科杂志,2008,44:244-247)
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