基底前脑巢蛋白阳性及阴性胆碱能神经元自然衰老及抗损伤的研究

发布时间：2018-07-28 17:57

【摘要】：研究背景基底前脑一般指位于大脑半球内侧面一些灰质结构,与其他脑区无明显界限。基底前脑是一个具有高度异质性的区域,含有多种不同化学属性的神经元,主要包括胆碱能神经元、谷氨酸能神经元以及r-氨基丁酸神经元等。最近研究发现成年大鼠和成人基底前脑存在着一群巢蛋白免疫阳性(nestin-ir)神经元,并证实这群神经元属于胆碱能神经元。进一步研究发现巢蛋白免疫阳性(nestin-ir)胆碱能神经元对学习记忆及对中枢神经系统的损伤与修复起重要作用。国内外初步研究提示相对于巢蛋白阴性胆碱能神经元,基底前脑的巢蛋白免疫阳性胆碱能神经元可能具有更强的抗损伤能力,表现出更好的神经可塑性。目的本课题采用基底前脑神经元原代培养方法,通过细胞免疫荧光染色研究巢蛋白免疫阳性及阴性胆碱能神经元抗衰老及抗氧化损伤能力的不同,进一步揭示两种神经元的神经可塑性的差异,为相关的神经系统疾病的治疗来提供新基础。方法实验采用出生24h以内的新生SD大鼠,每次用量8只左右,取其基底前脑处的神经元进行体外培养,培养周期11天,分别在培养的第1天,第3天,第5天,第7天,第9天和第11天,取培养的基底前脑神经元细胞爬片进行免疫荧光实验,研究巢蛋白免疫阳性及免疫阴性胆碱能神经元的自然衰老过程。抗损伤的研究采用出生24h以内的新生SD大鼠,每次用量8只左右,取其基底前脑部位的神经元进行体外培养,在培养的第3天时,放入150μmol/L H2O2构建细胞损伤模型,作用12h后,取出培养的基底前脑神经细胞爬片进行免疫荧光实验,观察两种神经元得抗损伤能力差异。结果体外培养第三天和第五天细胞生长达到高峰,这时神经元胞体较大,神经元突起粗大,部分神经元之间形成网状的突触连接。其中可见巢蛋白阳性神经元胞体较大,突起粗大且长;巢蛋白阴性神经元胞体小,突起较少。体外培养五天之后的神经元细胞数目逐渐减少,胞体饱和度降低。但巢蛋白阳性神经元数目下降速率缓慢,占细胞总数的比例逐渐增加,巢蛋白阴性神经元数目下降速率较快,所占细胞总数的比例逐渐下降。正常衰老组从培养第7天开始巢蛋白阳性及阴性胆碱能神经元数目差异明显,用T检验方法,P0.05,有统计学意义。这表明在相同的培养条件下,巢蛋白免疫阳性和阴性胆碱能神经元自然衰老和死亡速度是不同,巢蛋白免疫阳性胆碱能神经元细胞数目下降的速率比巢蛋白阴性胆碱能神经元慢。在神经元抗氧化损伤实验中,用T检验方法分析正常组和损伤组的巢蛋白阳性及阴性胆碱能神经元发现,正常组中两种神经元数目无明显差异,P0.05,无统计学意义。而损伤组中神经元P0.05,有统计学意义。这充分说明H2O2氧化损伤干预后巢蛋白阳性胆碱能神经元存活的数目较巢蛋白阴性胆碱能神经元多,细胞形态更好,比巢蛋白阴性胆碱能神经元有更强的抗损伤能力。结论体外培养的基底前脑神经元自然衰老及氧化损伤研究结果表明,与巢蛋白阴性胆碱能神经元相比,基底前脑的巢蛋白阳性胆碱能神经元抗衰老和抗损伤能力更强,表现出较强的神经可塑性。
[Abstract]:Background basal forebrain usually refers to some gray matter in the inner hemisphere of the cerebral hemisphere and has no distinct boundary with other brain regions. The basal forebrain is a highly heterogeneous region with a variety of neurons with different chemical properties, including cholinergic neurons, glutamic acid neurons and r- aminobutyric acid neurons. It was found that a group of nestin immunoreactive (Nestin-IR) neurons were found in the adult rat and adult basal forebrain, and confirmed that these neurons belong to cholinergic neurons. Further studies have found that nestin immunoreactive (Nestin-IR) cholinergic neurons play an important role in learning and memory and the damage and repair of the central nervous system. The preliminary study suggests that the nestin immunoreactive cholinergic neurons in the basal forebrain may have a stronger ability to resist damage and exhibit better neuroplasticity relative to nestin negative cholinergic neurons. The difference between the anti aging and antioxidant damage of the positive and negative cholinergic neurons further reveals the difference between the nerve plasticity of the two kinds of neurons and provides a new basis for the treatment of the related nervous system diseases. Methods the new SD rats under the birth 24h were used for each dose of about 8, and the nerve in the basal forebrain was taken. The cells were cultured in vitro and cultured for 11 days. The cultured basal forebrain neuron cells were taken for immunofluorescence test at first days, third days, fifth days, seventh days, Ninth days and eleventh days respectively, and the natural aging process of nestin immunoreactive and immune negative cholinergic neurons was studied. The anti injury study was born 24h. In the newborn SD rats, the neurons of the basal forebrain were cultured in vitro for about 8 rats each time. At the third day of the culture, 150 mu mol/L H2O2 was put into the cell damage model. After the action of 12h, the cultured basal forebrain nerve cell crawling slices were taken for immunofluorescence experiment, and the differences of the anti injury ability of the two kinds of neurons were observed. Results the cell growth reached the peak at third days and fifth days in vitro. At this time, the cell body was larger, the neurites were large, and the neurons formed a network of synapses. Among them, the nestin positive neurons were larger, the protuberances were large and long, and the nestin negative neurons were small, and the protuberances were less. In vitro culture, the cells were cultured for five days. The number of neuron cells decreased gradually and the cell saturation decreased, but the number of nestin positive neurons decreased slowly, the proportion of the total number of cells increased gradually, the number of nestin negative neurons decreased rapidly and the proportion of the total number of cells decreased gradually. The normal senescence group began to be nestin positive and negative from seventh days of culture. The difference of the number of sexual cholinergic neurons was obvious. The T test method, P0.05, had statistical significance. This showed that under the same culture conditions, the natural aging and death rate of nestin positive and negative cholinergic neurons were different, and the rate of nestin positive cholinergic neurons decreased more than the nestin negative cholinergic neurons. The T test was used to analyze the nestin positive and negative cholinergic neurons in the normal group and the injured group by T test. There was no significant difference in the number of neurons in the normal group, and there was no significant difference in the number of neurons in the normal group, P0.05, but the neuron P0.05 in the injured group was statistically significant. This fully explained the oxidative damage of H2O2. After intervention, the number of nestin positive cholinergic neurons survives more than nestin negative cholinergic neurons, and the cell morphology is better than nestin negative cholinergic neurons. Conclusion the results of natural senescence and oxidative damage of the basal forebrain neurons in vitro show that the nestin negative cholinergic neurons are the same as nestin negative cholinergic. In contrast, the nestin positive cholinergic neurons in basal forebrain were more resistant to aging and injury, showing stronger neuroplasticity.
【学位授予单位】：大理大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R741

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