【摘要】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nov mRNA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：nov mRNA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nov mRNA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：nov mRNA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nov mRNA,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。

DEVELOPMENT OF nov mRNA NEURONS IN CENTRAL NERVOUSSYSTEM OF THE RATIN SITU HYBRIDIZATION HISTOCHEMISTRY AND RT-PCR STUDY

Su Bingyin, Cai Wenqin^△，Zhang Chenggang, Perbal B^*

(Department of Histology and Embryology,The Third Military Medical University, Chongqing; ^*Université Paris Ⅶ, Paris, France)

【Abstract】Objective Examine the development of nephroblastoma overexpressed gene (nov) mRNA neurons in the central nervous system (CNS) of rats.MethodsIn 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.

【Key words】 Nephroblastoma overexpressed gene(nov); Central nervous system; Development; Rat

最近，人们发现了几种高度相关的基因，即结缔组织生长因子(connective tissue growth factor,CTGF)、cyr61和肾母细胞瘤过度表达基因(nephroblastoma overexpressed gene,nov)，它们合在一起称CCN家族，属于即刻－早期基因的一种类型，与c－fos等经典的即刻－早期基因不同^［1，2］。

nov基因是法国Perbal B等新近克隆出来的，由于在鸡肾母细胞瘤中高表达，故命名为肾母细胞瘤过度表达基因，随后的研究表明它是原癌基因的一种。编码产物(NOV蛋白)是一种胰岛素样生长因子(insulin－like growth factor,IGF)结合蛋白(IGF－binding protein,IGFBP),通过调节IGFs与其受体结合而发挥作用^［3～5］。用原位杂交组织化学方法证实在成年大鼠中枢神经系统中nov mRNA分布广泛，提示在大鼠中枢神经系统中nov基因可能起着重要的作用^［6］。为了探讨nov基因在整个个体发生过程包括老年阶段的表达变化，本实验拟用原位杂交组织化学和逆转录PCR方法对大鼠中枢神经系统nov mRNA神经元的发育进行系统研究，并进而探讨nov基因与神经系统发育的相关性及对神经系统可能的调控作用。

材料和方法

1. 试剂

限制性内切酶XbaI、地高辛标记试剂盒(SP6/T7)、抗地高辛抗体(工作浓度1∶1 000)、碱性磷酸酶显色剂NBT/BCIP(工作浓度1∶100)，PCR试剂盒均购自德国Boehringer Mannheim公司。

2. 材料

Wistar大鼠，体重200～230g，雌、雄分笼饲养数日，合笼后至检出阴栓为E0，出生时记为P0，用于PCR取材时直接断头，取大脑皮质，提取总RNA。原位杂交组织化学分别取P0～P5、P8、P15、P30、P60、P300，每组5只，断头取脑、脊髓于Zamboni液中固定10h，再浸入含30%蔗糖的Zamboni液中(4℃)至组织块沉入瓶底，30μm厚连续冰冻切片漂于Zamboni液中备用。

3. cRNA核酸探针的制备

含nov原癌基因重组质粒(pBX1.5)按氯化钙法程序操作^［7］。经宿主菌HB101(本室吴康博士惠赠)扩增，按碱裂解法分离纯化，所扩增质粒(长度4.5kb)经限制性内切酶XbaI酶切为3.5kb和1kb两个片断。回收含T7启动子的3.5kb片断，采用地高辛标记系统体外转录成含624nt的cRNA探针。

4. 原位杂交组织化学染色程序

用漂浮法进行nov mRNA原位杂交组织化学染色^［7］：0.1mol/L PBS(pH7.2)漂洗5min×3次，0.1mol/L 甘氨酸/PBS漂洗5min，0.3%Triton X-100/PBS漂洗25min，蛋白酶K(2mg/L)37℃孵育25min(于0.1mol/L Tris·5HCL pH8.0;0.05mol/L EDTA缓冲液中)，4%多聚甲醛中漂洗5min, 0.1mol/L PBS漂洗5min×3次，浸入新配制的0.1mol/L Tris·5HCL(pH8.0,含0.25%乙酸酐)5min，2×SSC 10min，切片在含0.5mg/L地高辛标记的nov cRNA探针的杂交液中42℃反应24h后，4×SSC中漂洗15min，2×SSC(含RNAase A 20mg/L)中漂洗30min(37℃)，1×SSC、0.5×SSC中各漂洗10min(37℃)，0.05mol/L PBS漂洗10min×2次，将切片转入碱性磷酸酶标记的抗地高辛抗体中孵育4h(37℃)，0.05mol/L PBS漂洗10min×3次，TSM1、TSM2中各漂洗10min×2次，继之用NBT/BCIP显色液显色^［7］，室温3～5h，然后裱片、脱水、DPX封片。

为了检验本实验的专一性和可靠性，作以下阴性对照。(1)将切片用RNA酶进行预处理后杂交；(2)以不含探针的杂交缓冲液取代含Dig-nov-cRNA探针的杂交液；(3)以正常羊血清代替羊抗Dig抗体-Ap复合物。

5.RT-PCR定量检测大鼠脑不同发育时期nov mRNA

分别取E18、P5、P15、P30、P60、P150和P300大鼠，每组3只。脑组织总RNA提取按文献方法进行^［8］，用1μg RNA加20μl的反应液(50mmol/L KCl,3mmol/L MgCl₂，10mmol/L Tris·5HCl pH8.3,1mmol/L DTT,5μmol/L随机引物，500μmol/L 4dNTP_S,26U RNASin,8U M-MLV反转录酶)，37℃ 1h后，100℃加热5min使逆转录酶失活。根据人nov基因第5外显子序列设计引物，上游：5′CTAAGCTTTAGTCTT CAG CTC CAG C 3′，下游：5′ CTGAATTCCTCAAAGCCATCC ACC 3′。PCR的反应总体积为30μl，含上述逆转录反应液2μl,200μmol/L dNTPs,10×Buffer 2.5μl，96.6℃ 3min预变性后，94℃ 60s,55℃ 60s,72℃ 60s循环30次，反应产物置2%琼脂糖凝胶电泳，摄片，扫描半定量。

结 果

1. 大鼠中枢神经系统nov mRNA神经