【作者】 肖飞；

【导师】 罗焕敏；

【作者基本信息】 暨南大学 ， 病理学与病理生理学， 2010， 博士

【摘要】 目的研究Nogo-66受体(Nogo-66 receptor, NgR)能否作为药物治疗阿尔茨海默病(Alzheimer’s disease, AD)的潜在靶标。此靶标是否具备促进皮质神经元突起再生和抑制β淀粉样蛋白(amyloid P-protein, Aβ)生成的双重作用。同时寻找具备促进皮质神经元突起再生和抑制Aβ生成双重作用的AD治疗药物。方法(1)体外无血清培养新生大鼠皮质神经元,采用神经元特异性烯醇化酶(neuron-specificenolase, NSE)和微管相关蛋白2(microtubule-associated protein2,MAP2)免疫化学染色法进行神经元鉴定；(2)采用不同细胞密度和不同浓度的B27培养大鼠皮质神经元,观察其对皮质神经元生长的影响；(3)体外培养大鼠皮质神经元,加入不同浓度的Nogo-66活性片段——Nogo-P4,用MTT法检测不同浓度的Nogo-P4 (3.5μM、7μM、14μM)对皮质神经元的毒性作用,通过细胞形态学观察,突起生长状况的定量分析,观察Nogo-P4对皮质神经元突起生长的影响,应用ELISA (enzyme-linked immunoadsordent assay)法测定细胞分泌的Aβ42含量,观察Nogo-P4对皮质神经元Aβ42分泌的影响；(4)体外培养大鼠皮质神经元,分别加入Nogo-66受体阻断剂NEP1-40、受体下游信号分子ROCK (rho-associated coiled coil-containing protein kinase)的抑制剂Y-27632和PKC (protein kinase C, PKC)抑制剂GO6976,采用上述指标,研究Nogo-P4影响皮质神经元突起生长和Aβ42分泌的机制；(5)采用Nogo-P4制作细胞模型并进行药物筛选,从中药单体中寻找有效化合物。结果(1)经两种不同的神经元特异性标志物(NSE和MAP-2)鉴定,确定所培养的细胞绝大多数为神经元；(2)合适的细胞密度(1200个／mm2)和合适的B27浓度(2%B27)有利于皮质神经元的生长,神经突起生长较长；(3)不同浓度的Nogo-P4对皮质神经元的存活率没有影响,不具有细胞毒作用。不同浓度的Nogo-P4能减少有突起细胞数量,明显降低神经元的平均突起长度,抑制皮质神经元突起的再生,同时还能增加培养基中的Aβ42含量,促进皮质神经元分泌Aβ42,并具有量效关系；(4)Nogo-66受体拮抗剂NEP1-40、PKC抑制剂GO6976和ROCK抑制剂Y-27632对正常皮质神经元的突起生长和Aβ42的分泌无影响,对Nogo-P4引起的皮质神经元突起再生抑制均有拮抗作用,能增加有突起细胞数量和神经元平均突起长度,对Nogo-P4引起的Aβ42生成有不同的影响。NEP1-40能促进皮质神经元突起再生,但不能减少Aβ42的生成。GO6976能促进皮质神经元突起再生,同时增加Aβ42的生成。Y-27632不仅能促进皮质神经元突起再生,同时也能减少Aβ42的生成；(5)经过药物筛选,找到一个有效单体SQ,能拮抗Nogo-P4增加有突起细胞数量和神经元平均突起长度,促进神经元突起的再生。但SQ不能减少皮质神经元分泌Aβ42。结论(1) Nogo-P4能抑制大鼠皮质神经元的突起再生,同时促进大鼠皮质神经元分泌Aβ42; (2)Nogo-P4抑制皮质神经元突起再生是通过Nogo-66受体及下游信号分子ROCK和PKC的激活所引起。其促进Aβ42的分泌与其受体下游信号分子ROCK的激活有关；(3) Nogo-66受体可以作为促进皮质神经元突起再生的靶标,不能作为抑制Aβ42生成的靶标,可以作为AD治疗的靶标,但不是最佳靶标。PKC可以作为促进皮质神经元突起再生的靶标,同时又是促进Aβ42生成的靶标,综合考虑,PKC不宜作为AD治疗的靶标。ROCK可以作为促进皮质神经元突起再生和抑制Aβ42生成双重作用的靶标,是药物治疗AD的一个理想靶标,针对此靶标的AD治疗药物将更加有效；(4)中药单体SQ能促进皮质神经元突起再生,但不能抑制Aβ42的生成,其可能的作用靶标是Nogo-66受体。更多还原

【Abstract】 ObjectivesTo find out whether Nogo-66 receptor has a dual role in the inhibition of amyloid P protein (A) generation and promotion of regeneration neurite, whether it can be a potential target for drug therapy of Alzheimer’s disease (AD). To search AD therapy drugs which have a dual role in inhibition of Aβproduction and promotion of neurite regeneration.Methods(1)The neurons from cerebral cortex of newborn rat were cultured in B27-supplemented Neurobasal, a serum-free medium combination, and identified by using NSE and MAP2 immunocytochemical staining.(2)The rat cortical neurons were cultured in different cell densities and different concentrations of B27,to observe its effect on neuron growth.(3)The rat cortical neurons were cultured with various concenstration (3.5μM,7μM,14μM) of Nogo-P4 (Nogo-66 receptor agonist).The cell viability of neuron was determined by the MTT method.To observe the effect of Nogo-P4 on the neurite growth, the convert phase microscope was used to analyse the number of cell with neurites, as well as the average length of neurite. To observe the effect of Nogo-P4 on AP42 secretion of cortical neurons, enzyme-linked immunoadsordent assay (ELISA) was carried out to analyze AP42 production. (4) The mechanism of Nogo-P4 on cortical neurite regeneration and Aβ42 secretion were studied by adding Nogo-66 receptor blocker NEP1-40 and receptor downstream signaling molecule ROCK inhibitor Y-27632 and PKC inhibitor GO6976.(5)We search the effective compounds from traditional Chinese medicine by using Nogo-P4 treated cell model for drug screening.Results(1)Most of the neurons were stained to be positive with two different neuron-specific markers NSE and MAP-2 immunocytochemical, suggesting the majority of cultured cells were neurons.(2)Appropriate cell density (1,200 cells/mm2) and appropriate concentration of B27 (2%B27) were beneficial on the growth of neurons.(3) The MTT assay showed that Nogo-P4 at different concentrations didn’t have any toxic effect on cortical neuron survival. Different concentraion of Nogo-P4 could reduce the number of cell with neurite and the average length of neurite.Moreover,Nogo-P4 significantly increased the production of Aβ42.(4)Nogo-66 receptor antagonists could promote neuronal regeneration, but could not reduce the generation of AP42.Use of PKC inhibitors could promote neuronal regeneration, and increased A042 generation.Use of ROCK inhibitors could not only promote neuron regeneration, but also reduce the generation of AP42.(5)Through drug screening, an effective compound SQ was found. SQ could antagonize Nogo-P4 to increase the number of cell with neurites and the average length of neurite. SQ did not reduce neuronal secretion of Aβ42.Conclusion(1) Nogo-P4 inhibits neurite regeneration of rat cortical neurons, while Nogo-P4 promotes Aβ42 secretion of rat cortical neurons.(2)Nogo-P4 inhibits neurite regeneration by directly activating Nogo-66 receptor and downstream signaling molecules ROCK and PKC.The Nogo-P4 promotes the Aβ42 secretion by directly activating downstream signaling molecules ROCK.(3)Nogo-66 receptor can be used as target promoting neurite regeneration, but not as a target for inhibition of Aβ42 generation, it can be used as target for AD drug therapy, but is not the best target. PKC can be used as target promoting neurite regeneration, but also to promote Aβ42 generation.So PKC is not appropriate to use as a target for AD drug therapy. ROCK is a target which can truly inhibit AP42 production and promote neurite regeneration, is an ideal drug target for AD therapy. Drugs targeting ROCK will be more effective. (4)SQ, the compound from traditional Chinese medicine, can promote neurite regeneration, but can not reduce AP42 production.The pharmacological target of SQ possibly is Nogo-66 receptor.更多还原