【作者】 闫鹏；

【导师】 杜怡峰；

【作者基本信息】 山东大学 ， 神经病学， 2009， 硕士

【摘要】 第一部分纤维化Aβ_1-40对体外培养胆碱能神经元活性及胰岛素信号传导通路相关蛋白表达影响的研究研究目的1、探讨纤维化Aβ_1-40对体外培养大鼠基底前脑胆碱能神经元活性及形态的影响;2、探讨纤维化Aβ_1-40对体外培养大鼠基底前脑胆碱能神经元胰岛素信号传导通路的影响;研究方法1、对Wistar胎鼠基底前脑胆碱能神经元进行体外原代培养,并以细胞免疫荧光的方法对所培养细胞进行鉴定;2、分别用0.1μmol/L、1μmol/L、2μmol/L、5μmol/L、10μmol/L五个终浓度的纤维化Aβ_1-40分别对原代培养的细胞进行干预,后应用MTT（四甲基偶氮唑盐）法检测细胞活性,并行细胞免疫荧光后于显微镜下观察其细胞形态变化;3、提取细胞总蛋白以免疫印迹的方法对Aβ_1-40干预前后神经元胰岛素信号传导通路相关蛋白进行检测。研究结果1、以0.1μmol/L和1μmol/L两个终浓度的Aβ_1-40进行干预的胆碱能神经元,其形态学无明显改变,MTT实验显示其细胞活力无明显改变;而以2μmol/L、5μmol/L和10μmol/L三个终浓度Aβ_1-40进行干预的胆碱能神经元,其细胞形态学观察及MTT实验均显示神经元活性有下降趋势,其下降程度与Aβ_1-40终浓度呈正相关,差异有统计学意义。以5μmol/L终浓度的Aβ_1-40对细胞进行不同时段（24h,48h,72h）干预后,细胞形态学观察及MTT实验均显示神经元活性的下降程度与Aβ_1-40的干预时间呈正相关,各组差异之间有统计学意义。2、Western Blot检测结果显示:以0.1μmol/L和1μmol/L两个终浓度的Aβ_1-40进行干预的胆碱能神经元,其胰岛素信号传导通路相关蛋白均无明显变化;而以2μmol/L、5μmol/L和10μmol/L三个终浓度Aβ_1-40进行干预的胆碱能神经元,其胰岛素信号传导通路相关蛋白中,InsR、IRS-1、Akt/PKB及Bcl-2的表达减低,减低程度与Aβ_1-40干预浓度呈正相关,差异有统计学意义;以5μmol/L终浓度的Aβ_1-40对细胞进行不同时段（24h,48h,72h）干预后,InsR、IRS-1、Akt/PKB及Bcl-2的表达减低与Aβ_1-40干预时间呈正相关;但未发现CREB表达的改变。研究结论Aβ_1-40对体外培养大鼠基底前脑胆碱能神经元的活性及胰岛素信号转导通路有损害作用,且与剂量呈正相关,提示在阿尔茨海默病的发病中纤维化Aβ_1-40可能通过对胰岛素信号传导通路的损害作用进而造成神经元活性的下降甚至凋亡、死亡丢失。第二部分阿尔茨海默病患者尿中AD7c-NTP含量的研究研究目的探讨中国老年人尿液中阿尔茨海默病相关的神经丝蛋白（AD7c-NTP）的含量在AD（阿尔茨海默病）临床诊断中的应用价值。研究方法以中国老年人为研究对象,采用酶联免疫吸附测定法（ELISA）,分别检测AD（阿尔茨海默病）患者组（分轻、中、重3组）65例、VaD（老年血管性痴呆）组60例、智能正常老年对照组120例尿液中AD7c-NTP的含量,所有数据均经SPSS13.0软件进行统计学处理。研究结果1、AD组、VaD组与智能正常老年对照组尿液中AD7c-NTP含量分别为（2.50±0.45）ng/mL、（1.19±0.68）ng/mL和（1.21±0.74）ng/mL,AD组明显高于另外两组,而VaD组与智能正常老年对照组之间比较,差异无统计学意义（P＞0.05）;2、阿尔茨海默病（AD）组内患者尿液AD7c-NTP含量与其痴呆程度呈正相关。研究结论尿液中AD7c-NTP的含量测定在阿尔茨海默病的诊断,尤其是早期诊断中有重要的临床参考价值。更多还原

【Abstract】 Part 1 Effects of fibrillar Aβ_（1-40） on the growth and the Insulin signal transduction pathway related protein of cultured primitive rat basal forebrain cholinergic neuronsObjective1.To investigate the affection of fibrillar Aβ_（1-40） to morphology and cellular vigor of cultured primitive rat basal forebrain cholinergic neurons in vitro;2.To investigate the affection of fibrillar Aβ_（1-40） to the insulin signal transduction pathway of cultured primitive rat basal forebrain cholinergic neurons in vitro.Methods1.Primitive rat basal forebrain neurons were cultured and evaluated with immuno-fluorescence;2.The cultured neurons were exposed to fibrillar Aβ_（1-40） with different ending concentrations（0.1μmol/L,1.0μmol/L,2.0μmol/L,5.0μmol/L & 10μmol/L） and different durations.Then morphologic and cellular vigor changes were observed by fluorescent microscope and MTT assay respectively;3.Investigate the expression of insulin signal transduction pathway related proteins of cultured neurons after the neurons were intervened by fibrillar Aβ_（1-40）.Results1.After being exposed to fibrillar Aβ_（1-40） for 48h,the neurons exposed to low ending concentration（0.1 & 1.0μmol/L） of Aβ_（1-40） showed no significant changes in morphology and cellular vigor;while the neurons exposed to higher ending concentration（2.0,5.0& 10.0μmol/L） of Aβ_（1-40） showed significant changes in morphology and cellular vigor: average process quantity of the neurons,average process length and the length of the longest process of neurons were decreased significantly,and the results of MTT assay showed that average OD values were significantly decreased.2.The expression of InsR、IRS-1、Akt/PKB、CREB and Bcl-2 in the cultured neurons showed no significant changes after being exposed to Aβ_（1-40） with the two lower concentrations（0.1 & 1.0μmol/L）;While after being exposed to the three higher concentrations（2.0,5.0&10.0μmol/L） of Aβ_（1-40）,the expression of InsR、IRS-1、Akt/PKB and Bcl-2 were significantly decreased and the expression of CREB showed no significant change.When the neurons were cultured with 5μmol/L Aβ_（1-40） for different duration（24h, 48h,72h）,the above indexes were decreased by time dependency except the expression of CREB.ConclusionsAβ_（1-40） have a concentration-dependent and time-dependent harmful effect on the activity and insulin signaling pathway of cultured primitive rat basal fore-brain cholinergic neurons, which suggest that the harmful effect to insulin signal transduction pathway induced by fibrillar Aβ_（1-40） may contribute to the apoptosis and loss of cholinergic neurons in Alzheimer’s disease.Part 2 Study of urinary AD7c-NTP levels in patients with Alzheimer’s diseaseObjectiveTo investigate the value of urinary AD7c-NTP assay in the clinical diagnosis of AD.MethodsWith the method of Enzyme-linked immunosorbent assay,to measure urinay AD7c-NTP levels in 245 elderly Chinese people,who were divided into three group including patients with AD（n=65）,patients with vascular dementia（VaD,n=60） and normal mental state elderly people（n=120）,.Statistical analysis was performed using SPSS program 13.0.Results1.The urinay AD7c-NTP assay of three groups were（2.50±0.45） ng/ml,（1.19±0.68）ng/ml and（1.21±0.74）ng/ml.As compared to normal mental state control and VaD group,there was a significant increase of urinary AD7c-NTP levels in AD group.No significant difference was found between VaD group and normal mental state control; 2.Within the AD group,AD7c-NTP level is positively correlated with the severity of the disease.ConclusionsThe measurement of urinary AD7c-NTP level is of important and practical value in the clinical diagnosis,especially early diagnosis,and Condition assessment of AD.更多还原