胱天蛋白酶3在啮齿类动物和猕猴脑中的表达:种系和年龄相关研究

背景:有研究显示,凋亡相关蛋白酶—胱天蛋白酶3被认为是动物和人脑中细胞凋亡过程中的关键酶,正常人或阿尔茨海默病患者的脑中有该酶的表达,这与对成年啮齿动物脑研究的结果之间存在着矛盾。目的:揭示胱天蛋白酶3在啮齿类动物和猕猴脑中的表达有无差异,分析该酶在猕猴脑中的表达水平有无年龄差异,以及不同猕猴脑区间有无差别。设计:单变量均数的平行比较。单位:解放军总医院肝胆外科研究所,香港中文大学解剖学系。材料:实验于2003-08/2005-02在解放军总医院肝胆外科研究所及香港中文大学解剖学系进行的,所用的2,12,24和48周龄(每周龄组n=5)SD大鼠、ICR小鼠和快速衰老小鼠(senescence-acceleratedmice,SAM),雌雄不限,由香港中文大学实验动物服务中心提供;健康雌性4岁和20岁猕猴各4只,均购自解放军总医院医学实验动物中心(SCXK-(京)2003-002)。以上动物均在无任何实验干预的标准条件下养殖。方法:①所有脑组织标本均从猕猴和啮齿动物中新鲜切取,制成匀浆后通过免疫印迹法检测啮齿类动物和猕猴脑组织中胱天蛋白酶3蛋白的表达;②用同样的方法比较两个年龄组猕猴的大脑皮质、海马和小脑中该蛋白的表达量,并通过免疫组织化学法显示胱天蛋白酶3在猕猴3个脑区中的细胞分布状态。主要观察指标:①胱天蛋白酶3在啮齿类动物和猕猴脑中的免疫印迹检测;②胱天蛋白酶3在猕猴3个脑区中的细胞分布状态。结果:①免疫印迹检测结果:胱天蛋白酶3在3种2周龄的鼠脑中表达模式相同,在其它年长鼠脑中未检测到该酶;胱天蛋白酶3在成年和老年猕猴脑中均有持续表达,表达量低于2周龄鼠。胱天蛋白酶3在猴脑中以酶原(Mr32000)的形式存在,未发现该酶在猕猴脑中的表达量有年龄和脑区差异。②免疫组织化学法显示结果:猕猴大脑皮质胱天蛋白酶3的免疫反应性很低,海马的CA1、CA3和CA4区可见低至中度阳性染色的锥体细胞,小脑皮质的少数浦肯野氏细胞呈强阳性染色。老年猕猴除运动皮质第V层偶见呈强阳性染色的大锥体细胞外,其余与成年猴无明显差别。结论:啮齿类动物成年后,脑中胱天蛋白酶3的表达迅速下降至测不出,而该酶在成年及老年猕猴脑中仍维持在一定水平,其表达量和阳性细胞的分布状态无明显的年龄差异。

Expression of caspase-3 in rodent and monkey brain: a species- and age-related study

BACKGROUND: Caspase-3 is well recognized as the key caspase carrying out apoptosis in animal and human brain. To date, a few studies revealed the expression of caspase-3 protein in brains of normal persons and Alzheimer patients but data obtained from rodents exhibited much discrepancy.OBJECTIVE: To investigate the different expression patterns of caspase-3in rodent and monkey brain, and the different expression of caspase-3 in different brain regions and during aging in monkeys.DESIGN: Parallel comparison between means of single variable.SETTING: Institute of Hepatobiliary Surgery, Chinese PLA General Hospital and Department of Anatomy, the Chinese University of Hong Kong.MATERIALS: The experiment was carried out from August, 2003 to February, 2005 in Institute of Hepatobiliary Surgery, Chinese PLA General Hospital and Department of Anatomy, the Chinese University of Hong Kong. Sprague Dawley rats, ICR mice and senescence-accelerated mice (SAM) with ages ranging from postnatal 2, 12, 24 to 48 weeks(n=5 for each age group of different rodents) were included in the present study. All of these animals were supplied by Laboratory Animal Services Center, the hinese University of Hong Kong. Totally 8 rhesus monkeys aged 4 years (n=4) or 20 years (n=4) were selected from the Laboratory Animal Center in Chinese PLA General Hospital SCXK-(Beijing)2003-002]. Both ro dents and monkeys were female and were raised under standard conditions without any experimental interventions. METHODS: ①Brain tissue samples were taken freshly from both rodents and monkeys and made into homogenate. The expression of caspase-3 pro tein in brains of both rodents and monkeys was investigated with im munoblot. ② The expression levels in monkey brains were exhibited quantitatively with the same method in three brain regions, such as the frontal cortex, hippocampus and cerebellar cortex, for the two age-groups. In vivo distribution patterns of caspase-3-immunoreactive cells were further presented in 3 brain regions of monkeys through immunohistochemistry. MAIN OUTCOME MEATURES: ①Detection of caspase-3 protein with immunoblot in the brain of rodents and monkeys; ② Distribution patterns of caspase-3-immunoreactive cells in 3 brain regions of monkeys. RESULTS: ① Result of detection with immunoblot: The same pattern of caspase-3 protein expression in brain of three kinds of 2-week-old rodents. But the expression was not seen in any other brains of older ages. Caspase 3 was expressed in a relatively high level inboth adult and aged monkey brains, and the amount did not attain to the level in 2-week-old rodents. Caspase-3 Was expressed in the pattern of zymogen (Mr 32 000). The ex pressions of caspase-3 in brains of monkey were not different in ages and brain regions. ②Result of Immunohistochemistry: It was showed that most neurons in the frontal cortex lack detectable caspase-3 immunoreactivity, whereas low to moderate caspase-3 immunostaining be found mainly in pyramidal cells in CA1, CA3 and CA4 subfields of hippocampus. And in the cerebellum, a small number of Purkinje cells were strongly stained in their cytosol and dendrites. Age-related expression pattern of caspase-3 were not found except that in the motor cortex of aged monkeys in which there were a limited number of large pyramidal cells in layer Ⅴ that were strongly stained with caspase-3 antibody.③ Immunoblot procedure revealed that the caspase-3 protein expressed in monkey brains is in the form of zymogen (Mr 32 000) and there is no significant difference in caspase-3 expression level as a function of either brain region or age of animals.CONCLUSION: Unlike rodents in which caspase-3 protein rapidly drops to an undetectable level since animals grow up, the primate expresses caspase-3 constitutively in brain until the late period of lifetime. But there are no significant brain region- or age-related differences in the protein levels in monkey brain.