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目的观察氯化铝(AlCl_3)对大鼠海马淀粉样前体蛋白(APP)启动子区甲基化及β淀粉样蛋白(Aβ)含量的影响。方法健康6月龄雄性SPF级SD大鼠40只,分成4组(正常对照组和AlCl_3低、中、高剂量组),每组10只,各组分别腹腔注射AlCl_30、10、20和30 mg/kg组,正常对照组注射相应体积生理盐水,连续5天,休息2天,共8周。甲基化特异性PCR法(MSP)检测海马APP启动子区的甲基化状态,实时荧光定量PCR(RT-PCR)测定APP mRNA表达水平,ELISA法测APP、Aβ_(42)蛋白表达量。结果随着AlCl_3浓度的增加,大鼠平均逃避潜伏期明显延长(P<0.05),AlCl_3中、高剂量组穿越平台的次数明显减少(P<0.05);AlCl_3染毒组海马APP启动子区甲基化检出率降低(χ~2=27.61,P<0.05),其中,高剂量组APP甲基化的检出率低于其余3组(P<0.01),APP甲基化的检出率随AlCl_3染毒剂量增加而降低的趋势(趋势χ~2=19.08,P<0.01)。各AlCl_3染毒组APP mRNA的表达水平高于生理盐水组(F=8.973,P<0.05)。与正常对照组比较,各AlCl_3染毒组大鼠海马APP、Aβ_(42)蛋白浓度均增高(F=11.14,P=0.032;F=17.82,P=0.018)。与低剂量组相比,中剂量组APP蛋白表达量增加,高剂量组APP、Aβ_(42)蛋白表达量增加,差异均有统计学意义(P<0.05)。Aβ_(42)蛋白免疫组化法检测结果示,中、高剂量组海马神经细胞内外出现棕黄色物质,考虑有Aβ的沉积。结论氯化铝染毒可引起大鼠海马APP启动子甲基化水平降低,进而影响APP mRNA表达水平增高,海马组织APP表达量增多,从而导致Aβ产生增多而沉积。
Objective To investigate the effect of aluminum chloride (AlCl_3) on the promoter methylation of amyloid precursor protein (APP) and β-amyloid protein (Aβ) in rat hippocampus. Methods Forty six-month-old male Sprague-Dawley (SD) SD rats were divided into 4 groups (normal control group and AlCl 3 low, medium and high dose groups) with 10 rats in each group. AlCl 3, 10, 20 and 30 mg / kg group, the normal control group injected the corresponding volume of saline for 5 consecutive days, resting for 2 days, a total of 8 weeks. The methylation status of APP promoter region in hippocampus was detected by methylation-specific PCR (MSP), APP mRNA expression was measured by real-time fluorescence quantitative PCR (RT-PCR) and APP and Aβ 42 protein expression by ELISA. Results With the increase of AlCl_3 concentration, the average escape latency of rats increased significantly (P <0.05), while the number of AlCl_3 middle and high dose groups decreased significantly (P <0.05) (Χ ~ 2 = 27.61, P <0.05). The detection rate of APP methylation in the high-dose group was lower than that in the other three groups (P <0.01). The detection rate of APP methylation AlCl_3 dose increased and decreased (trend χ ~ 2 = 19.08, P <0.01). The expression of APP mRNA in AlCl 3 -treated group was higher than that in saline group (F = 8.973, P <0.05). Compared with the normal control group, the concentrations of APP and Aβ_ (42) protein in hippocampus of AlCl_3 group were all increased (F = 11.14, P = 0.032; F = 17.82, P = 0.018). Compared with the low dose group, the expression of APP protein in medium dose group increased, while that of high dose group increased (P <0.05). The results of Aβ42 immunohistochemistry showed that there were brownish yellow substances inside and outside the hippocampal neurons in middle and high dose groups, considering the deposition of Aβ. Conclusion Aluminum chloride can cause the decrease of APP promoter methylation in hippocampus, which in turn increases the expression of APP mRNA and the expression of APP in hippocampus, resulting in the deposition of Aβ.