细胞外信号调节激酶系统 (ERKs)在正常发育大鼠视皮层基因表达的研究

INVESTIGATION OF TRANSCRIPTS OF ERK mRNAs IN DEVELOPMENTAL VISUAL CORTEX OF RATS

细胞外信号调节蛋白激酶 (ERKs)是皮层神经元生长、发育和分化的关键因子。本研究目的在于研究 ERKs(ERK1、ERK2和 ERK3 ) m RNA在视皮层各层的分布、表达量以及发育过程变化。实验用健康雄性 SD大鼠 ,于生后 (P) 14、2 1、2 8、45和 90 d(成年 )灌注固定 ,取全脑 ,切取视皮层。用 4%多聚甲醛固定 ,石蜡包埋 ,4μm厚切片。地高辛标记特异性寡核苷酸探针 (ERK1、ERK2 )和 c DNA探针 (ERK3 )。用原位杂交方法检测三种 ERKs亚型的 m RNA在各年龄组大鼠视皮层的表达。结果证明 :ERKs m RNA在出生后大鼠正常发育视皮层的表达 ,ERK1和 ERK2 m RNA的分布具有明显的层的特异性 ,表达于除 I层 (分子层 )之外的 II-VI层 ,ERK2较 ERK1m RNA的表达更广泛、信号密度更强。ERK1和 ERK2 m RNA的转录在发育敏感期增高 ,从 P2 1～P2 8逐渐增加 ,P45时达到高峰 ,到成年时降低为相当于 P2 1的水平。 ERK3 m RNA在大鼠出生后视皮层的信号表达强 ,比较恒定 ,无明显的层分布特异性。本研究结果提示 ,出生后正常大鼠发育期视皮层 ERK1和 ERK2的 m RNA表达呈上调趋势 ,而 ERK3 m RNA在大鼠出生后视皮层的表达量中等 ,比较恒定 ,缺乏发育性变化特点。表明 ERK1和

The extracellular signal regulated protein kinases, ERKs, play a critical role in neuron growth, neuronal plasticity, morphological differentiation and survival in brain. The aim of this investigation is to study the localization of ERK1, ERK2 and ERK3 and their mRNAs expression in visual cortex of normal rat from postnatal day 14 to adult. Sprague Dawley rats from P14, P21, P28, P45, P90 (adult) were perfused through heart with normal saline, followed by 4% paraformaldehyde in 0.1 mol/L phosphate buffer. The brains were post fixed overnight at 4℃ in 4% paraformaldehyde in 0.1 mol/L phosphate buffer. Blocks containing the occipital lobe were embedded in paraffin and sectioned serially at 4 μm in the frontal plane or in a plane parallel to the cortex of the occipital lobe. Sections were stored at 4℃ for in situ hybridization. Synthetic oligonucleotide probes have been used for detection of ERK1 and ERK2 mRNAs. A pair of primers were designed and reverse transcription polymers chain reaction (RT PCR) was used to obtain ERK3 cDNA segment from normal rat visual cortex as a probe of ERK3. In situ hybridization was carried out on tissue sections using three specific digoxigenin labeled probes to localize ERKs expression pattern and to identify ERK1, ERK2 and ERK3 mRNA in visual cortex. The result showed that the hybridization signals for ERK1 and ERK2 were found in layer II to layer VI, while no signal was seen in layer I. ERK2 mRNA was more widely distributed than that of ERK1 and the signal density for ERK2 was stronger than that for ERK1. Both ERK1 and ERK2 mRNA expression gradually increased during the critical period from P21 to P28 with a peak at P45. The expression declined to a level parallel to those at P21. In contrast to ERK1 and ERK2, the expression pattern of ERK3 mRNA was absent of obvious distinct layer distribution and expression signal remained relatively strong in visual cortex following birth. The results indicate that ERK1/ERK2 mRNA expression in the rat postnatal visual cortex is up regulated by development and ERK3 mRNA expression shows no obvious changes in visual cortex following birth. It suggests that ERK1 and ERK2 may be involved in the regulation of the developmental plasticity of visual cortex in rat during the critical period. The results may be useful in insight into molecular mechanism of development in visual cortex. (Figures 1,4,5 on plate 52)