發(fā)布時(shí)間：2018-06-16 00:19

  本文選題：視神經(jīng) + NgR��； 參考：《第三軍醫(yī)大學(xué)》2005年碩士論文

【摘要】： 視神經(jīng)損傷是臨床常見的眼外傷,迄今仍缺乏有效的治療手段。目前的研究證實(shí):視神經(jīng)再生失敗的原因除了與其自身再生潛力低下,損傷后營養(yǎng)因子的缺乏,膠質(zhì)瘢痕的形成等有關(guān)外,其所處的微環(huán)境中存在著神經(jīng)生長抑制因子也是一個(gè)重要因素。2002年研究發(fā)現(xiàn):少突膠質(zhì)細(xì)胞及其所形成的髓鞘中存在著的抑制蛋白——Nogo-66、MAG、OMgp,均通過NgR發(fā)揮作用,針對NgR的單克隆抗體可有效地促進(jìn)中樞神經(jīng)元軸突再生。NgR在中樞神經(jīng)系統(tǒng)中分布廣泛,灰質(zhì)中含量較高,大腦皮層、海馬、背側(cè)丘腦、小腦顆粒細(xì)胞等都有分布,但是,視神經(jīng)作為一種特殊的中樞神經(jīng),其中是否存在NgR的表達(dá)尚未見報(bào)道,同時(shí),能否利用RNA干擾技術(shù)來抑制NgR基因表達(dá),繼而阻斷幾種抑制因子(Nogo-66、MAG、OMgp)的作用,進(jìn)而促進(jìn)視神經(jīng)再生修復(fù)也未見報(bào)道。 本實(shí)驗(yàn)的目的:檢測NgR mRNA在視神經(jīng)的表達(dá)情況,觀察視神經(jīng)損傷后NgR mRNA的表達(dá)變化,構(gòu)建NgR mRNA特異的siRNA表達(dá)質(zhì)粒,為進(jìn)一步通過RNA干擾途徑來抑制NgR基因表達(dá),促進(jìn)視神經(jīng)再生研究奠定基礎(chǔ)。 方法:實(shí)驗(yàn)分為3個(gè)部分: (一).使用地高辛標(biāo)記的寡核苷酸探針通過原位雜交方法檢測視神經(jīng)中NgR mRNA的表達(dá)情況; (二).采用熒光定量PCR方法檢測視神經(jīng)損傷后NgR mRNA的表達(dá)變化;(三).構(gòu)建NgR mRNA的siRNA表達(dá)質(zhì)粒。 結(jié)果:1.原位雜交證實(shí)視神經(jīng)NgR mRNA表達(dá)陽性,對照實(shí)驗(yàn)呈陰性反應(yīng);2.熒光定量PCR顯示視神經(jīng)損傷后NgR mRNA表達(dá)無顯著性變化(P0.05);3.成功的構(gòu)建了兩個(gè)NgR特異的RNA干擾表達(dá)質(zhì)粒,為下一步實(shí)驗(yàn)奠定了基礎(chǔ)。 結(jié)論:視神經(jīng)存在NgR mRNA表達(dá),提示NgR介導(dǎo)的抑制途徑可能是視神經(jīng)損傷后難以再生的一個(gè)原因;視神經(jīng)鉗夾傷后NgR mRNA表達(dá)無顯著性變化,而我們的前期實(shí)驗(yàn)顯示視神經(jīng)鉗夾傷后Nogo-A mRNA的表達(dá)增高,配體和受體表達(dá)的不一致性,提示了Nogo-A除了抑制再生外,可能還有其它尚未闡明的功能,較之阻斷
[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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R779.1;R346

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 葉劍,王正國,朱佩芳,彭錫嘉;視神經(jīng)損傷后Nogo-A mRNA表達(dá)的變化[J];中華眼底病雜志;2003年04期

2 葉劍,王正國,朱佩芳;Nogo-A mRNA在大鼠神經(jīng)組織中的表達(dá)和定位[J];中華醫(yī)學(xué)雜志;2002年07期

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本文編號：2024315