【作者】 乔彤；

【导师】 胡义珍；

【作者基本信息】 华中科技大学 ， 眼科学， 2009， 博士

【摘要】 目的：建立稳定的SD大鼠的急性和慢性青光眼模型，检测大鼠视网膜上BDNF和slit₂mRNA的表达以及RGC凋亡的状况，利用RNAi干扰技术下调slit₂mRNA的表达，以期通过上调BDNF的途径降低RGC的凋亡达到视神经保护的目的。方法：①采用大鼠眼前房灌注生理盐水和不同大鼠视神经夹持时间来选择前房稳定的急性高眼压大鼠模型作为视神经保护的研究；②巩膜表面血管结扎联合角巩缘血管网电凝制作慢性高眼压大鼠模型；③造模成功后摘除眼球，固定包埋后切成5-μm的视网膜切片。免疫组织化学SP法检测大鼠视网膜上的BDNF的表达和核酸分子原位杂交法检测slit₂mRNA在大鼠视网膜上的表达；④slit₂SiRNA模型鼠的右眼玻璃体腔内，取大鼠眼球固定切片进行BDNF的免疫组化检查、TUNEL检查RGC的凋亡以及核酸分子原位杂交检测slit₂mRNA的表达。结果：①选用模型是选用20秒（s）的大鼠视神经夹持模型作为视神经保护研究的模型，不仅可以达到生理盐水前房灌注的急性高眼压模型的RGC凋亡水平，而且保持了眼球的完整性；②巩膜表面血管结扎联合角巩缘血管网电凝可以制作稳定的大鼠慢性高眼压模型。③BDNF的免疫染色见于节细胞层、内网层、内核层和视锥视杆层，其中视网膜节细胞层（GCL）可见大量胞浆深染的BDNF阳性细胞。Slit₂mRNA在视网膜RGC层和内核层均有表达，在急性和慢性高眼压模型的视网膜RGC层上的表达是上调的；④急性高眼压模型中和慢性高眼压模型中运用各浓度的slit₂mRNA玻璃体腔注射后视网膜上slit₂mRNA下调，同时BDNF的表达上调，RGC的凋亡减少，但没有显著性意义。而慢性高眼压模型中表现更为明显，在200nmol组中表现最为明显，并与其他组别有显著性差异。结论：在大鼠高眼压模型中，Slit₂siRNA注入玻璃体腔中可通过下调slit₂mRNA在视网膜节细胞层的表达，从而上调BDNF途径减少RGCs的凋亡，以达到保护视网膜视神经的作用，在慢性高眼压模型中更为有效。更多还原

【Abstract】 Objectives: To establish stable acute and chronic rat glaucoma models, clarify theexpression of BDNF and Slit₂mRNA in retina of rat glaucoma model, and observe theeffect of RNAi technique usage in retinal neuroprotection, we aim to upregulate theexpression of BDNF and reduce the apoptosis of RGC in retina by downregulating theexpression of slit₂mRNA in retina.Methods:①To select a stable rat nerve damage mode between high intraocularpressure model and rat nerve crush model;②To establish a rat chronic elevated intraocularpressure glaucoma model combined with ligation of episcleral veins and cauterization oflimbus arcades;③The eyes of rats after successful being made model were enucleated andprocessed for paraffin sectioning. Each eye was oriented so that the sections （5-μmthickness） were cut from the superior to the inferior edge. BDNF was traced byimmunohistochemistry SP method and Slit₂mRNA was evaluated by In situ hybridizationmethod;④we used 32 gauges needle to inject the given slit₂mRNA in differentconcentration into vitreous body of the acute and chronic rat glaucoma models. Eyes wereenucleated and processed for paraffin sectioning. Each eye was oriented so that the sections（5-μm thickness） were cut from the superior to the inferior edge. BDNF was traced byimmunohistochemistry SP method and Slit₂mRNA was evaluated by In situ hybridizationmethod and TUNEL assays were performed on 5-um slides of rat eye to detect the RGCapoptosis.Results①We selected the nerve crush 20s as the model for neuroprotection research;②we established a reliable glaucoma rat model by episcleral veins ligation combined withcauterization of limbus arcades;③Expressions of BDNF were detected in retinal ganglioncell layer and inner nuclear layer. In situ hybridization studies indicated that the slit₂mRNAwas specifically expressed in the RGC layer of rat retina and upregulated in the glaucoma rat model;④Different concentration of srSlit₂mRNA in acute and chronic rat model led todownreguiate Slit₂mRNA in retina and upregulate BDNF in retina and alleviate theapoptosis of RGCs, however, the outcomes were more abvious in chronic glaucoma model.Conclusions Introvitreal injection slit₂mRNA in glaucoma rat models could benefitfor neuroprotection through upregulating BDNF expression and downregulating theapoptosis of RGCs in the retina.更多还原