视神经NgR mRNA的表达及其siRNA表达质粒载体的构建

发布时间：2018-06-16 00:19

本文选题：视神经 + NgR　；参考：《第三军医大学》2005年硕士论文

【摘要】： 视神经损伤是临床常见的眼外伤,迄今仍缺乏有效的治疗手段。目前的研究证实:视神经再生失败的原因除了与其自身再生潜力低下,损伤后营养因子的缺乏,胶质瘢痕的形成等有关外,其所处的微环境中存在着神经生长抑制因子也是一个重要因素。2002年研究发现:少突胶质细胞及其所形成的髓鞘中存在着的抑制蛋白——Nogo-66、MAG、OMgp,均通过NgR发挥作用,针对NgR的单克隆抗体可有效地促进中枢神经元轴突再生。NgR在中枢神经系统中分布广泛,灰质中含量较高,大脑皮层、海马、背侧丘脑、小脑颗粒细胞等都有分布,但是,视神经作为一种特殊的中枢神经,其中是否存在NgR的表达尚未见报道,同时,能否利用RNA干扰技术来抑制NgR基因表达,继而阻断几种抑制因子(Nogo-66、MAG、OMgp)的作用,进而促进视神经再生修复也未见报道。本实验的目的:检测NgR mRNA在视神经的表达情况,观察视神经损伤后NgR mRNA的表达变化,构建NgR mRNA特异的siRNA表达质粒,为进一步通过RNA干扰途径来抑制NgR基因表达,促进视神经再生研究奠定基础。方法:实验分为3个部分: (一).使用地高辛标记的寡核苷酸探针通过原位杂交方法检测视神经中NgR mRNA的表达情况; (二).采用荧光定量PCR方法检测视神经损伤后NgR mRNA的表达变化;(三).构建NgR mRNA的siRNA表达质粒。结果:1.原位杂交证实视神经NgR mRNA表达阳性,对照实验呈阴性反应;2.荧光定量PCR显示视神经损伤后NgR mRNA表达无显著性变化(P0.05);3.成功的构建了两个NgR特异的RNA干扰表达质粒,为下一步实验奠定了基础。结论:视神经存在NgR mRNA表达,提示NgR介导的抑制途径可能是视神经损伤后难以再生的一个原因;视神经钳夹伤后NgR mRNA表达无显著性变化,而我们的前期实验显示视神经钳夹伤后Nogo-A mRNA的表达增高,配体和受体表达的不一致性,提示了Nogo-A除了抑制再生外,可能还有其它尚未阐明的功能,较之阻断
[Abstract]:Optic nerve injury is a common ocular injury in clinic, so far, there is still a lack of effective treatment. Current studies have confirmed that the failure of optic nerve regeneration is related to its low regeneration potential, lack of nutritional factors after injury, glial scar formation, and so on. The presence of nerve growth inhibitor in the microenvironment is also an important factor. In 2002, it was found that the inhibitory protein Nogo-6OMgp in oligodendrocytes and the myelin sheath formed by oligodendrocytes, all act through NgR. The monoclonal antibody against NgR can effectively promote axon regeneration of central neurons. NgR is widely distributed in the central nervous system, and the content of gray matter is higher. The cerebral cortex, hippocampus, dorsal thalamus and cerebellar granulosa cells are all distributed. As a special kind of central nervous system, the expression of NgR in optic nerve has not been reported. At the same time, can RNA interference technology be used to inhibit the expression of NgR gene, and then block the effect of several inhibitory factors, such as Nogo-6nMAGOMgp. Furthermore, the promotion of optic nerve regeneration and repair has not been reported. The purpose of this study was to detect the expression of NgR mRNA in optic nerve, observe the change of NgR mRNA expression after optic nerve injury, and construct a siRNA expression plasmid specific to NgR mRNA, so as to further inhibit the expression of NgR gene by RNA interference pathway. The research of promoting optic nerve regeneration lay the foundation. Methods: the experiment was divided into three parts: (1). The expression of NgR mRNA in optic nerve was detected by in situ hybridization with digoxigenin labeled oligonucleotide probe. Fluorescence quantitative PCR was used to detect the expression of NgR mRNA after optic nerve injury. The siRNA expression plasmid of NgR mRNA was constructed. The result is 1: 1. The expression of NgR mRNA in optic nerve was positive by in situ hybridization, and negative reaction was found in control experiment. Fluorescence quantitative PCR showed no significant change in the expression of NgR mRNA after optic nerve injury. Two NgR specific RNAi expression plasmids were successfully constructed, which laid a foundation for further experiments. Conclusion: there is NgR mRNA expression in optic nerve, which suggests that the inhibition pathway mediated by NgR may be one of the reasons why it is difficult to regenerate after optic nerve injury, but the expression of NgR mRNA does not change significantly after optic nerve clamp injury. Our previous experiment showed that the expression of Nogo-A mRNA increased and the expression of ligand and receptor was not consistent after optic nerve clamp injury, suggesting that Nogo-A may have other unelucidated functions in addition to inhibiting regeneration.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R779.1;R346

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