本文選題：SMS2 + 自噬��； 參考：《河南大學(xué)》2011年碩士論文

【摘要】：自噬是存在于真核細(xì)胞中普遍的生命現(xiàn)象,是近十年來生命科學(xué)領(lǐng)域一個新的研究熱點,在降解胞內(nèi)蛋白、蛋白質(zhì)結(jié)構(gòu)重建、清除廢物、保持細(xì)胞內(nèi)環(huán)境穩(wěn)定等方面發(fā)揮著非常重要的作用。自噬對于降解錯誤折疊和聚集蛋白方面起著關(guān)鍵的作用,與神經(jīng)變性疾病關(guān)系密切,受到越來越多研究者的關(guān)注。本課題以SMS2基因敲除(SMS2~(-/-))小鼠為研究對象,應(yīng)用MAPLC3和Beclin-1免疫細(xì)胞化學(xué),PCR、電鏡和Western blotting等技術(shù)方法,以期探討SMS2基因敲除小鼠海馬神經(jīng)細(xì)胞內(nèi)一種重要的調(diào)節(jié)細(xì)胞凋亡的第二信使-神經(jīng)酰胺增多后,對于神經(jīng)細(xì)胞自噬現(xiàn)象的影響,為通過誘導(dǎo)自噬治療神經(jīng)變性疾病提供臨床治療依據(jù)。 為了研究SMS2基因敲除小鼠海馬神經(jīng)細(xì)胞內(nèi)神經(jīng)酰胺增多后,對于神經(jīng)細(xì)胞自噬現(xiàn)象的影響,本實驗室從美國紐約州立大學(xué)動物實驗中心引進(jìn)的SMS2基因敲除小鼠的雜合子SMS2~(+/-)小鼠為種鼠,利用PCR方法鑒定培育出的SMS2~(-/-)小鼠為模型,探討神經(jīng)酰胺對神經(jīng)細(xì)胞自噬的影響,從而為神經(jīng)變性疾病的臨床治療和和預(yù)防提供理論依據(jù)。 目的:利用SMS2基因敲除小鼠探討神經(jīng)酰胺對海馬神經(jīng)細(xì)胞自噬現(xiàn)象的影響,從而為神經(jīng)變性疾病的臨床治療和和預(yù)防提供理論依據(jù)。 方法:利用從美國紐約州立大學(xué)動物實驗中心引進(jìn)的SMS2~(+/-)小鼠(雄性一只,雌性兩只,背景為C57BL/6J小鼠)為種鼠,將雜合子SMS2~(+/-)小鼠采用雜交、回交、互交的方法進(jìn)行繁殖,用酚氯仿提取法提取小鼠基因組DNA, PCR擴(kuò)增目的基因,瓊脂糖凝膠電泳對小鼠基因型做出鑒定,獲得雄性和雌性純合子SMS2~(-/-)小鼠,按照同樣方法進(jìn)行繁殖;以SMS2~(-/-)小鼠為研究對象,野生型(WT)小鼠為對照組,利用透射電子顯微鏡觀察P14和P30模型組與對照組海馬神經(jīng)細(xì)胞中自噬和自噬溶酶體結(jié)構(gòu);利用免疫熒光染色技術(shù)觀察P7、P14和P30模型組和對照組神經(jīng)細(xì)胞自噬細(xì)胞數(shù),并觀察Beclin-1與MAPLC3免疫細(xì)胞化學(xué)染色在神經(jīng)細(xì)胞的共表達(dá)情況;利用Western blotting方法檢測P14和P30模型組與對照組小鼠海馬CA1區(qū)神經(jīng)細(xì)胞MAPLC3的蛋白水平;采用單因素方差分析LSD檢驗對數(shù)據(jù)進(jìn)行分析。 結(jié)果: 1. SMS2~(-/-)小鼠海馬CA1區(qū)神經(jīng)細(xì)胞自噬現(xiàn)象:①電子顯微鏡下,SMS2~(-/-)小鼠較WT小鼠海馬神經(jīng)細(xì)胞內(nèi)出現(xiàn)較多自噬體或自噬溶酶體樣結(jié)構(gòu);②光鏡下,SMS2~(-/-)小鼠P7、P14和P30 CA1區(qū)神經(jīng)細(xì)胞自噬細(xì)胞數(shù)較WT小鼠高,變化有顯著差異(P㩳0.01)。2. Beclin-1對于自噬的調(diào)節(jié)作用:①Beclin-1在SMS2~(-/-)小鼠與WT小鼠的表達(dá)情況比較與MAPLC3基本一致,P7、P14和P30 SMS2~(-/-)小鼠Beclin-1陽性細(xì)胞數(shù)明顯多于對照組(P0.01)。②Beclin-1與MAPLC3二者在同一細(xì)胞中基本呈重疊表達(dá)3.利用Western blotting檢測P14和P30 SMS2~(-/-)小鼠和WT小鼠海馬CA1區(qū)神經(jīng)細(xì)胞MAPLC3蛋白的相對表達(dá)量,SMS2~(-/-)小鼠較WT小鼠高,變化有顯著差異(P㩳0.01)。 結(jié)論:SMS2~(-/-)小鼠海馬神經(jīng)細(xì)胞內(nèi)神經(jīng)酰胺增多,神經(jīng)酰胺不僅促進(jìn)細(xì)胞的凋亡,同時促進(jìn)自噬,造成小鼠海馬神經(jīng)細(xì)胞自噬現(xiàn)象增強(qiáng)。
[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.
【學(xué)位授予單位】：河南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R346

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 石淵淵;李志強(qiáng);谷敬麗;王玉蘭;;神經(jīng)鞘磷脂合成酶基因沉默對細(xì)胞凋亡的影響[J];高等學(xué)�；瘜W(xué)學(xué)報;2009年09期

2 鄧錦波，蔡琰，，邱建勇，鞠躬，戴洪，孫曉江，王s

本文編號：1982533