SMS2基因敲除小鼠海马神经细胞自噬现象的研究

发布时间：2018-06-05 15:50

本文选题：SMS2 + 自噬　；参考：《河南大学》2011年硕士论文

【摘要】：自噬是存在于真核细胞中普遍的生命现象,是近十年来生命科学领域一个新的研究热点,在降解胞内蛋白、蛋白质结构重建、清除废物、保持细胞内环境稳定等方面发挥着非常重要的作用。自噬对于降解错误折叠和聚集蛋白方面起着关键的作用,与神经变性疾病关系密切,受到越来越多研究者的关注。本课题以SMS2基因敲除(SMS2~(-/-))小鼠为研究对象,应用MAPLC3和Beclin-1免疫细胞化学,PCR、电镜和Western blotting等技术方法,以期探讨SMS2基因敲除小鼠海马神经细胞内一种重要的调节细胞凋亡的第二信使-神经酰胺增多后,对于神经细胞自噬现象的影响,为通过诱导自噬治疗神经变性疾病提供临床治疗依据。为了研究SMS2基因敲除小鼠海马神经细胞内神经酰胺增多后,对于神经细胞自噬现象的影响,本实验室从美国纽约州立大学动物实验中心引进的SMS2基因敲除小鼠的杂合子SMS2~(+/-)小鼠为种鼠,利用PCR方法鉴定培育出的SMS2~(-/-)小鼠为模型,探讨神经酰胺对神经细胞自噬的影响,从而为神经变性疾病的临床治疗和和预防提供理论依据。目的:利用SMS2基因敲除小鼠探讨神经酰胺对海马神经细胞自噬现象的影响,从而为神经变性疾病的临床治疗和和预防提供理论依据。方法:利用从美国纽约州立大学动物实验中心引进的SMS2~(+/-)小鼠(雄性一只,雌性两只,背景为C57BL/6J小鼠)为种鼠,将杂合子SMS2~(+/-)小鼠采用杂交、回交、互交的方法进行繁殖,用酚氯仿提取法提取小鼠基因组DNA, PCR扩增目的基因,琼脂糖凝胶电泳对小鼠基因型做出鉴定,获得雄性和雌性纯合子SMS2~(-/-)小鼠,按照同样方法进行繁殖;以SMS2~(-/-)小鼠为研究对象,野生型(WT)小鼠为对照组,利用透射电子显微镜观察P14和P30模型组与对照组海马神经细胞中自噬和自噬溶酶体结构;利用免疫荧光染色技术观察P7、P14和P30模型组和对照组神经细胞自噬细胞数,并观察Beclin-1与MAPLC3免疫细胞化学染色在神经细胞的共表达情况;利用Western blotting方法检测P14和P30模型组与对照组小鼠海马CA1区神经细胞MAPLC3的蛋白水平;采用单因素方差分析LSD检验对数据进行分析。结果: 1. SMS2~(-/-)小鼠海马CA1区神经细胞自噬现象:①电子显微镜下,SMS2~(-/-)小鼠较WT小鼠海马神经细胞内出现较多自噬体或自噬溶酶体样结构;②光镜下,SMS2~(-/-)小鼠P7、P14和P30 CA1区神经细胞自噬细胞数较WT小鼠高,变化有显著差异(P㩳0.01)。2. Beclin-1对于自噬的调节作用:①Beclin-1在SMS2~(-/-)小鼠与WT小鼠的表达情况比较与MAPLC3基本一致,P7、P14和P30 SMS2~(-/-)小鼠Beclin-1阳性细胞数明显多于对照组(P0.01)。②Beclin-1与MAPLC3二者在同一细胞中基本呈重叠表达3.利用Western blotting检测P14和P30 SMS2~(-/-)小鼠和WT小鼠海马CA1区神经细胞MAPLC3蛋白的相对表达量,SMS2~(-/-)小鼠较WT小鼠高,变化有显著差异(P㩳0.01)。结论:SMS2~(-/-)小鼠海马神经细胞内神经酰胺增多,神经酰胺不仅促进细胞的凋亡,同时促进自噬,造成小鼠海马神经细胞自噬现象增强。
[Abstract]:Autophagy is a universal life phenomenon in eukaryotic cells. It is a new research hotspot in the field of life science in the past ten years. It plays a very important role in the degradation of intracellular protein, protein structure reconstruction, cleaning of waste, and maintaining the stability of the intracellular environment. Autophagy plays a key role in the degradation of wrong folding and aggregation protein. The role of the bond is closely related to neurodegenerative diseases and is concerned by more and more researchers. This subject uses the SMS2 gene knockout (SMS2~ / -)) mice as the research object, using MAPLC3 and Beclin-1 immunocytochemistry, PCR, electron microscopy and Western blotting, in order to explore a kind of weight in the hippocampal neurons of SMS2 knockout mice. The effect of the second messenger - ceramide on the phenomenon of autophagy after regulating cell apoptosis provides a clinical basis for the treatment of neurodegenerative diseases by inducing autophagy.
In order to study the effect of the neuroamide increase in the hippocampal neurons of SMS2 gene knockout mice, the SMS2 gene knockout mouse SMS2~ (+ / -) mouse was introduced from the animal experiment center of New York State University, United States, and the SMS2~ (- / -) mouse was identified by PCR method. To investigate the effect of ceramide on autophagy of neural cells, so as to provide a theoretical basis for clinical treatment and prevention of neurodegenerative diseases.
Objective: To explore the effect of ceramide on the autophagy of hippocampal neurons by using SMS2 gene knockout mice, so as to provide a theoretical basis for the clinical treatment and prevention of neurodegenerative diseases.
Methods: SMS2~ (+ / -) mice, introduced from the animal experiment center of New York State University, USA (male one, two female, and background C57BL/6J mice) were used as mice. The heterozygote SMS2~ (+ / -) mice were bred by hybridization, backcross and cross interbreeding. The genomic DNA of mice was extracted by phenol chloroform extraction, and the target gene was amplified by PCR, agarose. The mouse genotypes were identified by gel electrophoresis. The male and female homozygote SMS2~ (- / -) mice were obtained and propagated according to the same method. The SMS2~ (- / -) mice were used as the research object and the wild type (WT) mice as the control group. The autophagy and autophagosome knot in the hippocampus neurons of the P14 and P30 model group and the control group were observed by transmission electron microscope. The number of autophagic cells in P7, P14 and P30 model groups and the control group were observed by immunofluorescence staining, and the co expression of Beclin-1 and MAPLC3 immunocytochemical staining in the nerve cells was observed, and Western blotting method was used to detect the MAPLC3 protein water in P14 and P30 model groups and the hippocampus CA1 region of the control mice. The data were analyzed by one-way ANOVA LSD test.
Results: 1. SMS2~ (- / -) hippocampal CA1 autophagy in the hippocampus of mice: 1. Under electron microscope, more autophagosome or autophagosome like structure appeared in SMS2~ (- / -) mice than that of WT mice. The number of autophagic cells in SMS2~ (- / -) mice P7, P14 and P30 CA1 region neurons was higher than that of WT mice (P? 0.) 01) the regulating effect of.2. Beclin-1 on autophagy: (1) the expression of Beclin-1 in SMS2~ (- / -) mice and WT mice was basically the same as that of MAPLC3. The number of Beclin-1 positive cells in P7, P14 and P30 SMS2~ (- / -) mice was obviously more than that of the control group (P0.01). The relative expression of MAPLC3 protein in the hippocampal CA1 region of P14 and P30 SMS2~ (- / -) mice and WT mice was detected. The SMS2~ (- / -) mice were higher than those of WT mice, and the changes were significantly different (P? 0.01).
Conclusion: the neuroamide in the hippocampal neurons of SMS2~ (- / -) mice increased, and ceramide not only promoted the apoptosis of the cells, but also promoted autophagy, which resulted in the enhancement of autophagy in the hippocampal neurons of mice.
【学位授予单位】：河南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346

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