【作者】 吕丹丹；

【导师】 宋焱峰； 景玉宏；

【作者基本信息】 兰州大学 ， 人体解剖与组织胚胎学， 2011， 硕士

【摘要】 目的以糖尿病动物为模型研究持续高血糖,高血脂对认知功能,海马神经细胞损伤及其突触可塑性的影响。通过体外细胞模型,分别研究高糖及高脂对PC12细胞的损伤效应,这一损伤效应与细胞增殖以及自噬的关系。方法1、C57B/L小鼠按体重连续给予四天腹腔注STZ,持续高脂饮食。对照组给予等量的无菌柠檬酸/柠檬酸钠缓冲液腹腔注射,普通饲料喂养。一周后,从鼠尾采血分离血浆,测定葡萄糖浓度,血糖≥200mg/dL作为模型成功的标志。模型小鼠高脂饮食喂养一个月后做胰岛素敏感性实验。随后每个月小鼠尾部采血并采用葡萄糖氧化酶法连续监测各组小鼠血糖变化,采用酶比色法连续监测各组小鼠血液中甘油三酯及胆固醇变化,通过水迷宫实验检测动物学习记忆变化,通过Fluoro-Jade-C染色观察并计数海马区细胞密度及退变的神经细胞,并用免疫组织化学染色、ELISA法检测海马区β-淀粉样蛋白含量,通过Golgi-Cox染色结合体视学半定量技术分析海马CA1区锥体细胞树突棘密度变化,通过透射电子显微镜观察海马CA1区神经细胞自噬情况。2、用葡萄糖体外培养PC12细胞,应用MTT法加入不同浓度葡萄糖以及不同作用时间检测细胞存活率,并确定合适的损伤模型；普通显微镜下观察细胞形态并进行细胞计数,Brdu掺入后采用免疫荧光显微技术观察细胞增殖的情况；采用流式细胞术测定细胞周期及增殖情况。用油酸诱导体外培养的PC12细胞,应用MTT法加入不同浓度油酸作用不同时间检测细胞存活率,并用Western blotting检测不同浓度油酸对细胞LC3-Ⅱ表达水平的影响,加入自噬抑制剂3-MA及自噬诱导剂雷帕霉素应用Western blotting检测处理后细胞内LC3-Ⅱ的表达水平,采用MTT法检测细胞存活率,应用MDC染色观察自噬囊泡。结果1、体内实验结果表明,2型糖尿病小鼠在一个月出现胰岛素抵抗,并伴随持续的高血糖及高胆固醇血症。2个月,4个月出现学习记忆能力下降(p<0.05)。通过Fluoro-Jade-C染色表明4个月糖尿病小鼠海马区退变的神经细胞数量增加。通过ELISA及免疫组化观察到β淀粉样蛋白表达增加。利用Golgi-Cox染色观察到海马CA1区锥体细胞树突棘密度显著下降(p<0.01)。2、体外实验结果表明,随着葡萄糖浓度增加,细胞存活率逐渐下降,葡萄糖作用于PC12细胞48h,其有效致死量为150mM/L,选取此浓度建立损伤模型,显微镜下观察高糖作用组细胞数量与正常组比较明显减少(p<0.01)。Brdu掺入实验发现高糖抑制PC12细胞增殖。通过流式细胞仪检测细胞周期,表明高糖作用48h后PC12细胞被阻滞在G1期,S期细胞数量减少。给予不同浓度油酸可以诱导PC12细胞出现自噬,自噬相关基因LC3-Ⅱ表达水平明显升高,MDC染色荧光显微镜观察可见大量自噬泡出现。自噬抑制剂3-MA导致LC3-Ⅱ表达下调,同时细胞存活率下降。雷帕霉素促进LC3-Ⅱ的表达,同时也促进了细胞的存活。结论1、体内研究结果表明2型糖尿病持续的高血糖、高血脂可能直接导致海马区神经细胞损伤,β-样淀粉蛋白沉积,突触可塑性下降,上述变化导致认知功能障碍。2、高糖诱导PC12细胞后形态没有明显的变化,但增殖受到抑制。高脂能够诱导自噬的出现,给予自噬抑制剂后细胞损伤严重,而给予自噬诱导剂雷帕霉素后有保护效应。这说明高脂诱导的自噬活化是一种保护性代偿效应。更多还原

【Abstract】 ObjectiveIn vivo, our study focuses on the effects of hyperglycemia and hyperlipidemia on cognition, neural degeneration and synaptic plasticity in hippocampus. In vitro, our experiments study the changes of proliferation and autophagy on PC12 cell with high glucose or high oleic acid, respectively.Methods1. C57B/L mice were injected with STZ (30mg/kg, i.p) once a day for four days consecutively. The control mice were injected with the equal volume of sterile citric acid/sodium citrate buffer. After one week, blood plasma was harvest from tail vein, and the concentration of glucose was determined, and≥250mg/dL was used. Plasma glucose was measured by oxidase-methods and plasma cholesterol was test by Enzyme assay every month. Learning and memory was test using the moires water maze. And neural degeneration in hippocampus was valued through Fluoro-Jade C-staining.β- amyloid deposition in hippocampus was measured by ELISA and immunohistochemistry. Golgi-Cox staining together with semi-quantitative stereological method was used to detect the density of dendritic spine of pyramidal cells in CA 1 of hippocampus. Additionally, autophagosome of pyramidal cells in hippocampus were observed by transmission electron microscopy.2. PC12 cells were cultured at different condition to mimicry high glucose or high lipid by adding the different concentration of glucose or oleic acid to medium. Cell viability was valuated by MTT assay. Cell morphology were observed by microscope and cell proliferation by immunofluorescence microscope with high glucose; Also cell cycle was determined by flow cytometry. Under high lipid condition, presenting of 3-MA, autophagy inhibitor, or rapamycin, autophagy inducer, respectively, the expression level of LC3-Ⅱwere detected by western blotting, cell viability were valuated by MTT.Results1. In vivo, our results indicates that serum insulin levels were lower and plasma insulin sensitivity were decreased in diabetic mouse compared with control group at 4 weeks after induced. The ability of learning and memory declined compared to the age-matched group after 2 mouth (p<0.05). Neural degeneration in hippocampus aggregated at 4 month compared with that of control. More deposition ofβamyloid in hippocampus of DM-mouse exits than control. The density of dendritic spine in hippocampus is lowed in DM-mouse compared to the control group (p< 0.01). Autophagysome were observed in cytoplasma of pyramidal cells in hippocampus in diabetic mouse by transmission electron microscopy.2. In vitro, the viability of PC 12 cell was obviously inhibited with different concentrations of glucose in time- and dose-dependent manners, half of inhibition concentration(IC50) value was 150mM. The proliferation of PC 12 cells significantly were inhibited with high glucose by incorperation of Brdu. Most of cells were arrested at G1 phase, and S phase synthesis of were reduced after 48h with high glucose. Autophagy were induced in PC 12 cells with oleic acid, and the expression levels of autopha related protein LC3-Ⅱwere significantly increased and autophagy vesica was observed using fluorescent microscope. Giving autophagy inhibitors. MTT show the cell vibility and the expression of LC3-Ⅱlevels decreased significantly compared with single oleic acid, while the opposite effect of rapamycin.Conclusion1. Our study suggested persistant high glucose and lipid induce the neurodegeneration and synapse-loss,enhancement of beta amyloid deposition in hippocampus, which impaired the cognition in type 2 diabetes model.2. PC 12 cells proliferation were inhibited by high glucose, autophagy was induced by oleic acid, also inhibit autophaby influence cell viability, but has protective effect of rapamycin.更多还原