[β-淀粉样肽31-35和25-35对培养的大鼠皮层神经元的神经毒性]

[Neurotoxicity of beta-amyloid peptide 31-35 and 25-35 to cultured rat cortical neurons].

作者信息

Zhang Xiao-hong, Yu Huan-ling, Xiao Rong, Xiang Li, Li Li, Ma Wei-wei, Zhang Jie, Chu Jin-hua

机构信息

School of Public Health and Family Medicine, Capital Medical University, Beijing 100069, China.

出版信息

Zhonghua Yu Fang Yi Xue Za Zhi. 2009 Dec;43(12):1081-5.

PMID:20193504

Abstract

OBJECTIVE

To compare the toxicity of mechanism beta amyloid peptide (Abeta) 25-35 and 31-35 to cultured rat cortical neurons.

METHODS

The primary rat cerebral cortical neurons of rat were cultured 48 hours and randomly divided into control, Abeta25-35 (25 micromol/L)and Abeta31-35 (25 micromol/L) treated groups. After twenty four hours culturing, the cells were collected MTT assay was performed to measure the viability of cultured neurons. The mitochondrial membrane potential was determined to investigate the alteration of mitochondrial structure and function of neurons by laser scanning confocal microscope. The DNA damage of neurons was measured by single cell gel electrophoresis. The expressions of Bcl-2, Bax and p53 gene were measured by reverse transcriptase polymerase chain reaction (RT-PCR). Each experiment was repeated three times.

RESULTS

The absorbance (0.746 +/- 0.071, 0.811 +/- 0.083) and fluorescence intensity (3.050 +/- 0.240, 2.806 +/- 0.203) of neurons in Abeta25-35 and 31-35 treated group were significantly lower (t(A) were 4.023 and 5.401, t(fluorescence intensity) were 9.524 and 7.589 respectively, P < 0.01) than those in control group (1.038 +/- 0.125 and 4.280 +/- 0.358 respectively). The percentage of comet cells (59.0%, 48.5%) and tail length (57.3 +/- 4.7, 54.2 +/- 6.8) microm in Abeta25-35 and 31-35 treated group were significantly higher (chi(2)(comet cell) were 99.397 and 137.071, t(tail length) were 19.058 and 29.173 respectively, P < 0.01) than those in control group (4.5% and (5.2 +/- 1.1) microm respectively). Compared with control group (Bax/Bcl-2 ratio 0.2090 +/- 0.0991, p53/beta-actin ratio 1.6560 +/- 0.0853), the Bax/Bcl-2 ratio (t value were 2.429 and 2.356 respectively, P < 0.05) and expressions of p53 (t value were 2.366 and 2.503 respectively, P < 0.05) gene were significantly increased (P < 0.05) in Abeta25-35 (Bax/Bcl-2 ratio 1.2774 +/- 0.0762, p53/beta-actin ratio 2.0284 +/- 0.2223) and Abeta31-35 (Bax/Bcl-2 ratio 1.0330 +/- 0.0683, p53/beta-actin ratio 1.9505 +/- 0.2725) treated group.

CONCLUSIONS

Neurotoxicity-induced by Abeta31-35 in cortical neurons is similar to that induced by Abeta25-35, which is possibly related to its direct neurotoxic and apoptotic effects to neurons.

摘要

目的

比较β淀粉样肽（Abeta）25 - 35和31 - 35对培养的大鼠皮质神经元的毒性。

方法

将原代培养48小时的大鼠大脑皮质神经元随机分为对照组、Abeta25 - 35（25微摩尔/升）处理组和Abeta31 - 35（25微摩尔/升）处理组。培养24小时后，收集细胞，采用MTT法检测培养神经元的活力。用激光扫描共聚焦显微镜测定线粒体膜电位，以研究神经元线粒体结构和功能的变化。通过单细胞凝胶电泳检测神经元的DNA损伤。采用逆转录聚合酶链反应（RT - PCR）检测Bcl - 2、Bax和p53基因的表达。每个实验重复3次。

结果

Abeta25 - 35和31 - 35处理组神经元的吸光度（0.746±0.071，0.811±0.083）和荧光强度（3.050±0.240，2.806±0.203）显著低于对照组（分别为1.038±0.125和4.280±0.358）（t（吸光度）分别为4.023和5.401，t（荧光强度）分别为9.524和7.589，P < 0.01）。Abeta25 - 35和31 - 35处理组彗星细胞百分比（59.0%，48.5%）和彗尾长度（57.3±4.7，54.2±6.8）微米显著高于对照组（分别为4.5%和（5.2±1.1）微米）（χ²（彗星细胞）分别为99.397和137.071，t（彗尾长度）分别为19.058和29.173，P < 0.01）。与对照组（Bax/Bcl - 2比值0.2090±0.0991，p53/β - 肌动蛋白比值1.6560±0.0853）相比，Abeta25 - 35（Bax/Bcl - 2比值1.2774±0.0762，p53/β - 肌动蛋白比值2.0284±0.2223）和Abeta31 - 35（Bax/Bcl - 2比值1.0330±0.0683，p53/β - 肌动蛋白比值1.9505±0.2725）处理组的Bax/Bcl - 2比值（t值分别为2.429和2.356，P < 0.05）和p53基因表达（t值分别为2.366和2.503，P < 0.05）显著升高（P < 0.05）。