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背景:糖尿病大鼠存在学习和记忆障碍,β淀粉样前体蛋白17肽具有改善这一行为的作用。糖尿病是否通过影响海马区神经细胞的膜电位及凋亡导致学习和记忆障碍以及β淀粉样前体蛋白17肽的相关作用机制尚不清楚。目的:观察糖尿病大鼠海马区神经细胞的线粒体膜电位和凋亡以及β淀粉样肽前体蛋白干预对其影响。设计:完全随机分组设计,对照实验。单位:首都医科大学宣武医院北京脑老化研究实验室和河北保定市第一中心医院内分泌科。材料:实验中指标测定部分于2002-05/2002-08在解放军总医院仪器测试中心完成。动物造模及干预阶段在首都医科大学附属宣武医院动物室完成。选用Wistar雄性大鼠18只。将大鼠按随机抽签法分为3组,正常对照组,糖尿病模型组,β淀粉样前体蛋白17肽治疗组,每组6只。方法:①糖尿病模型组和β淀粉样前体蛋白17肽治疗组大鼠禁食12h后用pH值4.4链脲佐菌素按60mg/kg腹腔注射诱发糖尿病模型。3d后测尾静脉血糖>15mmol/L为造模成功。正常对照组大鼠不造模。β淀粉样前体蛋白17肽治疗组于背部皮下注射β淀粉样前体蛋白17肽,3.4μg/只,每周3次,共10周。正常对照组及糖尿病组给予等量的生理盐水相同部位注射,每周3次,共10周。②满10周时,将大鼠麻醉,断头取脑后冰浴下取海马组织制备单细胞悬液,采用线粒体膜电位特异性荧光探针JC-1标记线粒体及Annexin-V-FITC&PI双染色技术,进行流式细胞仪上机检测线粒体膜电位和凋亡发生率。③数据间差异比较采用单因素方差分析。主要观察指标:各组大鼠海马神经细胞线粒体膜电位和海马单细胞凋亡率比较结果。结果:进入结果分析大鼠18只,每组6只。①海马神经细胞线粒体膜电位:糖尿病模型组明显低于正常对照组[(551.91±53.36),(809.88±82.41)道,P<0.01],β淀粉样前体蛋白17肽治疗组高于糖尿病模型组[(705.99±89.92)道,P<0.05],与正常对照组相近(P=0.146)。②海马单细胞凋亡率:糖尿病模型组明显高于正常对照组和β淀粉样前体蛋白17肽治疗组[(5.32±1.37)%,(1.03±0.55)%,(2.80±0.92)%,P<0.01,0.05]。结论:海马线粒体膜电位下降和细胞的凋亡可能参与糖尿病脑病的发生发展;β淀粉样前体蛋白17肽具有改善这一病理过程的作用。
BACKGROUND: Diabetic rats have learning and memory impairments and beta amyloid precursor protein 17 peptide has the effect of ameliorating this behavior. Whether diabetes mellitus affects learning and memory impairment through affecting the membrane potential and apoptosis of neurons in the hippocampus and the mechanism by which beta amyloid precursor protein 17 peptide is involved is unclear. OBJECTIVE: To observe the mitochondrial membrane potential and apoptosis of neurons in hippocampus of diabetic rats and the effect of β-amyloid precursor protein on them. Design: Complete randomized block design, control experiment. SETTING: Beijing Brain Aging Research Laboratory, Xuanwu Hospital, Capital Medical University, and Department of Endocrinology, First Central Hospital, Baoding, Hebei. Materials: The experimental part of the indicator determination was performed at the Instrument Testing Center of Chinese PLA General Hospital from May 2002 to August 2002. Animal modeling and intervention phase in the Capital Medical University Xuanwu Hospital animal room completed. 18 Wistar male rats were used. The rats were randomly divided into three groups according to random sampling method, normal control group, diabetic model group and β amyloid precursor protein 17 peptide treatment group, 6 rats in each group. Methods: ① Diabetic model group and β amyloid precursor protein 17 peptide-treated group were fasted for 12 h and then diabetic rats were induced by intraperitoneal injection of streptozotocin at a dose of 4.4 mg / kg. 3d measured tail vein glucose> 15mmol / L for modeling success. Normal control rats are not model. The amyloid precursor protein 17 peptide treatment group was subcutaneously injected β amyloid precursor protein 17 peptide (3.4 μg / mouse) three times a week for 10 weeks. Normal control group and diabetic group were given the same amount of normal saline injection, 3 times a week for 10 weeks. (2) After 10 weeks, the rats were anesthetized, the hippocampus was removed after decapitation and the brain was removed by ice bath, and the single cell suspension was prepared. The mitochondria and Annexin-V-FITC & PI double staining were performed using mitochondrial membrane potential-specific fluorescent probe JC- Technology, flow cytometry on the machine to detect mitochondrial membrane potential and the incidence of apoptosis. ③ Comparison of data using single-factor analysis of variance. MAIN OUTCOME MEASURES: Mitochondrial membrane potential and apoptosis rate of hippocampal single cells in hippocampal neurons of rats in each group were compared. Results: The results of entering the analysis of 18 rats, 6 in each group. ① Mitochondrial membrane potential of hippocampal neurons: The diabetic model group was significantly lower than that of the normal control group [(551.91 ± 53.36), (809.88 ± 82.41), P <0.01]; the amyloid precursor protein 17 peptide treatment group was higher than the diabetic model Group [(705.99 ± 89.92), P <0.05], similar to the normal control group (P = 0.146). ② The rate of single cell apoptosis in the hippocampus: Compared with the normal control group and the amyloid precursor protein 17 peptide-treated group [(5.32 ± 1.37)%, (1.03 ± 0.55)%, (2.80 ± 0.92)%, P <0.01, 0.05]. CONCLUSION: The decrease of mitochondrial membrane potential and cell apoptosis in hippocampus may be involved in the development of diabetic encephalopathy. The β amyloid precursor protein 17 peptide can improve the pathological process.