【作者】 周颖；

【导师】 乔中东；

【作者基本信息】 上海交通大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 α7烟碱型乙酰胆碱受体不仅在神经细胞上表达,而且在非神经细胞,例如内皮细胞、巨噬细胞、上皮细胞和肿瘤细胞等均有分布表达,参与这些细胞的功能调节,并与一些疾病的病理生理变化相关。将α7烟碱型乙酰胆碱受体作为靶点开发的受体激动剂已用于多种疾病的治疗。研究目的:流行病学研究表明吸烟能导致多种心血管疾病,尼古丁作为香烟中主要有毒成分在其中起着重要的作用,但其作用机制并不十分清楚。同时,尼古丁在阿尔茨海默病中起到保护神经细胞和提高认知能力的作用。尼古丁作为α7烟碱型乙酰胆碱受体激动剂,可能是通过激活受体发挥作用。本研究目的是探讨尼古丁通过直接、间接刺激对脐带静脉血管内皮细胞表达粘附分子的影响及其作用机制,然后探讨gx-50—一种通过药物软件分析筛选获得的特异性α7烟碱型乙酰胆碱受体激动剂对淀粉样蛋白诱导的大鼠皮质神经细胞损伤的影响。研究方法:首先,采用不同浓度的尼古丁刺激巨噬细胞（Ana-1）,在各时间点收集巨噬细胞培养上清测定上清中各细胞因子含量。并选择6×10^-5M,12小时处理组Ana-1细胞上清处理脐带静脉内皮细胞,收集各时间点内皮细胞的上清后检测其中的可溶性粘附分子。并用抗TNF-α,抗IL-1β,抗IFN-γ,抗TNF-α+抗IL-1β的单克隆抗体中和尼古丁处理的巨噬细胞上清液中相应的细胞因子,再观察其对血管内皮细胞粘附分子表达以及对白细胞粘附能力的影响。其次,用不同浓度尼古丁刺激血管内皮细胞,收集细胞上清用酶联免疫吸附法检测前列腺素E2（PGE2）的表达。并提取细胞RNA与蛋白对环氧合酶2（COX-2）的表达进行检测。然后用α7烟碱型乙酰胆碱受体拮抗剂银环蛇毒素和NF-κB抑制剂二硫代氨基甲酸吡咯烷（PDTC）预处理后观察COX-2和PGE2的表达,并用凝胶迁移实验检测尼古丁与抑制剂处理后NF-κB的转录水平变化。最后用COX-2抑制剂NS-398预处理细胞后观察尼古丁诱导的细胞间粘附分子-1蛋白表达变化。最后,用不同浓度的gx-50处理大鼠皮质神经细胞,MTT法检测其对细胞活性的影响。用MTT和PI/Hoechst33258双染法观察gx-50预处理后对淀粉样蛋白诱导皮质神经细胞损伤的影响,用实时定量PCR和Western blot法检测相关凋亡基因的表达。最后用受体竞争性结合实验检测gx-50与α7乙酰胆碱受体的结合能力。结果:IL-1β在6×10^-5M尼古丁处理后12小时达高峰,TNF-α在24小时达高峰。IL-8和IFN-γ的表达不受尼古丁的影响。用6×10^-5M的尼古丁处理Ana-1细胞12小时的上清刺激血管内皮细胞,能上调sICAM-1、sVCAM-1和E-selectin的表达。抗TNF-α,抗IL-1β,抗TNF-α+抗IL-1β单克隆抗体中和Ana-1上清中的相应细胞因子后,内皮细胞表达sICAM-1和sVCAM-1的量明显降低,且可以阻断单个核细胞与血管内皮细胞的粘附。此外,尼古丁可以浓度依赖性地增加COX-2基因表达和前列腺素E2的分泌,同时在乙酰胆碱受体拮抗剂银环蛇毒素和NF-κB抑制剂PDTC预处理后,这种表达增加明显得到了抑制。而且用COX-2抑制剂NS-398预处理后,尼古丁诱导的ICAM-1表达也得到了抑制。在gx-50实验中,结果显示1×10-7～1×10^-5M的gx-50对皮质神经细胞活性没有显著的影响。在1×10^-6的gx-50预处理后,淀粉样蛋白诱导的神经细胞凋亡数量明显下降,并且使Bcl-2/Bax比率显著上升。另外,gx-50能够结合到大鼠皮质神经细胞表面的α7烟碱型乙酰胆碱受体。结论:综上所述,尼古丁作为α7烟碱型乙酰胆碱受体激动剂可以通过巨噬细胞分泌炎性因子TNF-α,IL-1β间接诱导内皮细胞表达粘附分子ICAM-1和VCAM-1。同时,尼古丁还可以通过α7乙酰胆碱受体及NF-κB途径直接刺激内皮细胞表达炎性因子,并诱导内皮细胞表达ICAM-1。此外,gx-50作为一种潜在的α7烟碱型乙酰胆碱受体激动剂能够有效地抑制Aβ25-35诱导的皮质神经细胞凋亡,gx-50是否是通过α7烟碱型乙酰胆碱受体介导的神经保护机制仍然需要进一步的研究证实。更多还原

【Abstract】 Recent studies found that nicotinic acetylcholine receptors were expressed not only in neuron cells, but also in non-neuron cells, such as endothelial cells, macrophages, epithelial cells and tumor cells. The expression of nicotinic acetylcholine receptor is involved in regulating the function of these cells and related to pathological and physiological changes in some diseases.α7 nicotinic acetylcholine receptor agonists has been used to treat many diseases.Object:Firstly, the aim of our study is to investigate the indirect and direct effect of nicotine on the expression of adhesion molecules in human umbilical vein endothelial cells （HUVECs） and analyze the pathway mediated by nicotinic acetylcholine receptor.Scecondly, gx-50, an ingredient from Zanthoxylum, has been suggested to effectively active alpha7 neuronal nicotinic acetylcholine receptor by structure-based drug design methods. In the present study, we investigated whether alpha7 neuronal nicotinic acetylcholine receptor was involved in the neuroprotective effect of gx-50 on beta-amyloid （Aβ）25-35-induced neuronal damage in primary cultured cortical neurons. Methods:According to the concentration of nicotine in the serum of smokers, Ana-1 （macrophages） was challenged by different concentration nicotine, the supernatant of Ana-1 cells were collected for 3 h, 6 h, 12 h, 24 h and 48 h. The expression of IFN-γ,TNF-α,IL-1β,IL-8 were detected by RIA or enzyme-linked immunosorbent assay （ELISA）. Treatment of HUVECs with supernatant from Ana-1 stimulated by 6×10^-5 M nicotine for 12 h, the adhesion molecules were determined by ELISA. The supernatant from Ana-1 challenged by nicotine was pre-neutralized with anti-TNF-α, anti-IL-1β, anti-IFN-γ, or anti-TNF-α+ anti-IL-1βantibody together, respectively. The value of adhesion molecules was detected with ELISA. Adherent cells were counted in 5 microscopic high power fields.HUVECSs were treated by different concentration of nicotine. At the end of the incubation time, cell supernatants were collected. Levels of PGE2 were determined using enzyme immunoassay kit. The level of COX-2 mRNA and protein were detected by Real time-PCR and Western blot. Then, the cells were pre-incubated with 1×10^-6Mα-Bungarotoxin （BTX） or 5×10^-5 M PDTC for 30 min prior to 1×10^-6 M nicotine exposure, then the level of COX-2 mRNA and protein were measured by Real time-PCR and Western blot. The level of PGE2 was determined by ELISA. The effect of nicotine on NF-κB DNA binding activity is measured by EMSA. After NS-398 treatment, nicotine-induced ICAM-1 protein expression was measured by Western blot and Immunofluoresence staining analyses.Cortical neurons were treated with different concentration of gx-50 for 48 h, the viability were measured by MTT assay. The inhibitory effect of gx-50 on Aβ25-35-induced neurons cytotoxicity and apoptosis, MTT assay and PI/Hoechst 33258 dual staining method were used. Cells were incubated with 10-6 M gx-50 for 2 h then exposed to 2.5×10^-5 M Aβ25-35 for 48 h. The expression of anti-apoptotic gene Bcl-2 and pro-apoptotic gene Bax was detected by Real-time PCR and Western blot. The competitive binding assay was performed to verify gx-50 as an agonist ofα7 nAChR.Results:The expression of TNF-αand IL-1βin Ana-1 was improved as the increasing of nicotine concentration till 6×10^-5 M. Moreover, there was an expression peak of TNF-αat 24 h as well as IL-1βat 12 h. However, nicotine could not regularly affect the release of IFN-γand IL-8. The expression of sICAM-1 and sVCAM-1 were significantly decreased in the groups of anti-TNF-α, anti-IL-1βor anti-TNF-α+anti-IL-1βpre-neutralized, and blocked partly the attachment of mononuclear cells to HUVECs. TNF-αor IL-1βrelease could be affected by nicotine with time- and concentration-dependent manner.Nicotine increased COX-2 mRNA expression in a dose-dependent manner. The maximal level of COX-2 mRNA and protein were expressed after exposure to 1×10^-6 M nicotine for 2 h or 6 h. It was found thatα-bungarotoxin significantly decreased the level of COX-2 mRNA and protein by about 61.3% and 70.2% respectively of the nicotine-treated group. In addition, PDTC prevented the nicotine-induced COX-2 mRNA and protein expression by 73.5% and 80.1%. Nicotine-induced ICAM-1 protein expression was remarkably decreased after NS-398 pretreatment.Pretreatment of the cells with gx-50 for 2 h prior to 2.5×10^-5 M Aβ25-35 exposure caused significantly apoptotic rate decrease and viability elevation. Furthermore, a marked reduction of bcl-2/bax ratio was found after 2.5×10^-5 M Aβ25-35 exposure, which could be partly reversed by pretreatment of gx-50.Conclusion:Nicotine could increase the expression of ICAM-1 and VCAM-1 mediating TNF-α,IL-1βrelease by macrophages. In addition,nicotine induced COX-2 expression through NF-κB activation which mediated by nicotinic acetylcholine receptor and the induction of COX-2 was related to ICAM-1 expression. Furthermore, nicotine could induce COX-2 expression through activating nicotinic acetylcholine receptor and NF-κB pathway which was related to ICAM-1 expression. In addition, our research revealed that gx-50, an potentialα7 nAChR agonist could protect neurons against amyloid-induced neurotoxicity.更多还原