大鼠中枢神经系统肾母细胞瘤过度表达基因mRNA神经元的发育——原位杂交与RT-PCR研究

DEVELOPMENT OF nov mRNA NEURONS IN CENTRAL NERVOUS SYSTEM OF THE RAT IN SITU HYBRIDIZATION HISTOCHEMISTRY AND RT PCR STUDY

目的研究大鼠ＣＮＳ肾母细胞瘤过度表达基因（ｎｏｖ）ｍＲＮＡ神经元的发育。方法原位杂交组织化学和逆转录ＰＣＲ。结果Ｐ０～Ｐ４：原位杂交方法最早检测到ｎｏｖｍＲＮＡ神经元是Ｐ０，Ｐ０～Ｐ４阳性信号均较弱，分布于脑桥和脊髓等。Ｐ５：丘脑部分核团和海马锥体细胞层观察到阳性神经元。Ｐ８：ｎｏｖｍＲＮＡ神经元数目增多，边缘系统及丘脑明显阳性；大脑皮质和下丘脑阳性较弱。Ｐ１５：丘脑、下丘脑和大脑皮质ｎｏｖｍＲＮＡ神经元增多，其余核团稳定在Ｐ８水平。Ｐ３０：在脑干观察到强烈的阳性信号，大脑皮质强阳性，海马、丘脑和下丘脑标记较强。Ｐ６０：脑桥和延髓的标记仍然很强，大脑皮质、丘脑和下丘脑的阳性信号稳定在Ｐ３０水平。Ｐ３００：ｎｏｖｍＲＮＡ神经元显著减少，阳性信号明显减弱。逆转录ＰＣＲ检测结果与原位杂交所得结果基本相符，在出生前２ｄ检测到ｎｏｖｍＲ－ＮＡ，其表达高峰出现在Ｐ３０～Ｐ６０之间，随之下降，Ｐ１５０和Ｐ３００表达弱。结论ｎｏｖ基因与其他早期反应基因不同，其表达高峰不是在胚胎发育早期，而是在近性成熟时期，提示ｎｏｖ基因主要在神经系统发育、分化及功能活动中起作用。

Objective\ Examine the development of nephroblastoma overexpressed gene (nov) mRNA neurons in the central nervous system (CNS) of rats. Methods In situ hibridization histochemistry (ISHH) using digoxigenin labeled cRNA probes and reverse transcription polymerase chain reaction (RT PCR). Results\ In P0 P4,neurons expressing nov mRNA were detected firstly on P0 by ISHH. The hybridization signal was very weak in P0 P4, mainly distributed in facial nucleus, trigeminal nucleus, ventral and dorsal horn of spinal cord. On P5, positive neurons were found in some nucleus of thalamus and pyramidal layer of hippocampus. The number of nov mRNA neurons increased on P8. The positive neurons widely distributed in limbic system including hippocampus, amygdaloid nucleus, globus pallidus and lateral septal nucleus. The hybridization signal was strong in ventral posteromedial thalamic nucleus and ventral posterolateral thalamic nucleus, while they were weak in cerebral cortex and hypothalamus.On P15,the number of nov mRNA neurons increased in mediodorsal thalamic nucleus,posterior thalamic nucleus,dorsal hypothalamic nucleus,ventromedial hypothalamic nucleus,paraventricular hypothalamic nucleus and cerebral cortex.On P30,the strongest positive signal was found in facial nucleus,a group of vestibular nucleus,dorsal and ventral cochlear nucleus.Stronger positive signal was also observed in cerebral cortex.Hippocampus,thalamus and hypothalamus showed middle intensity of hybridization signal.On P60,the positive signal remained strong in pone and medulla.Stronger positive signal was detected in giganocellular reticular nucleus,suprafacial nucleus and dorsal cochlear nucleus.The positive signal in cerebral cortex,thalamus and hypothalamus was equal to the level of P30,on P300,the number of nov mRNA neurons reduced significantly,and the intensity of the positive signal was decreased remarkably,especially in cerebral cortex,hippocampus,amygdaloid nucleus and globus pallidus.The results of reverse transcription polymerase chain reaction correlated well with the results of in situ hybridization histochemistry.nov mRNA was detected on E18.The peak of expression appeared during P30 P60,and reduced gradually on P150 and P300.Conclusion\ Our results show that nov gene is different from the other immediate early genes.The peak of expression is not at early stage of embryo,but during P30 P60,suggesting that nov gene may play an important role in differentiationof of CNS and maintain the function of CNS.