[嗅神经离断后嗅上皮上气味受体基因的表达]

Liu Yue-hong, Wei Yong-xiang, Yang Ling, Miao Xu-tao, Lu Ya-yan, Fan Er-zhong, Liu Xiao-chao, Zhang Jing-feng

Department of Otorhinolaryngology Head and Neck Surgery, Beijing Tongren Hospital Affiliated of Capital Medical University, Beijing 100730, China.

Zhonghua Er Bi Yan Hou Tou Jing Wai Ke Za Zhi. 2009 Aug;44(8):669-74.

OBJECTIVE

To constitute the animal model of unilateral olfactory nerve transection and observe the expression level and distribution of odorant receptors.

METHODS

Thirty-two rats were divided into two groups: the olfactory nerve transection group (20) and the control group (12). The former group received the operation to transect the left olfactory nerve following the left olfactory bulb was exposed under microscope and the latter group did not give any disposal. At every stage of five days, two weeks, four weeks and six weeks after the operation, five rats from the nerve transection group and three from the control group were anaesthetized simultaneously, and olfactory epithelium were taken out after transcardial perfusion, then paraffin imbedding. Coronal sections were sliced for HE staining to observe the thickness changes of the olfactory epithelium, and for in situ hybridization (ISHs) to investigate the expression of olfactory receptor genes (Olr287, Olr226, Olr1493 and Olr1654) in the epithelium, also to evaluate the changes of the expression level and location of the selected receptors during the regeneration of olfactory epithelium.

RESULTS

HE staining showed that 5 days after the operation cell quantity and thickness of the olfactory epithelium decreased obviously, which increased gradually 2 or 4 weeks after operation. After 6 weeks' recovery, the thickness of the epithelium could reach the control level. The pattern of cell staining by ISH showed a specific spatial distribution along the anteroposterior (AP) and dorsoventral (DV) axis. Evidence suggested that odorant receptors were distributed in continuous and multiple overlapping bands in the normal or nerve transected-recovered epithelium rather than in the conventionally accepted three or four zones. The data also demonstrated that the distribution of sensory neuron types, as identified and defined by odorant receptor expression, was restored to normal or nearly so by 6 weeks after operation. Likewise, the numbers of probe-labeled neurons in the nerve transected-recovered had an obvious decrease 5 days after olfactory nerve transection. Reactive cells (x(-) +/- s) of Olr1493 in the operated side was (53.9 +/- 19.9), compared with (419.0 +/- 21.2) in the unoperated side, there was statistic significance between them (t = 63.960, P < 0.01). Reactive cells increased gradually according to the regeneration of the epithelium, and were nearly equivalent to the normal side 6 weeks later without significant differentiation (t = 2.600, P > 0.05), according to the absolute positive cells in the operated and unoperated side of (417.8 +/- 32.4) and (445.3 +/- 10.0) respectively.

CONCLUSION

The regeneration of the sensory neurons and receptors, both the number and the distribution, can recover to normal after olfactory nerve transection.

目的

构建单侧嗅神经切断的动物模型，观察气味受体的表达水平及分布情况。

方法

将32只大鼠分为两组：嗅神经切断组（20只）和对照组（12只）。前者在显微镜下暴露左侧嗅球后进行切断左侧嗅神经的手术，后者不做任何处理。术后5天、2周、4周和6周的每个阶段，同时麻醉嗅神经切断组的5只大鼠和对照组的3只大鼠，经心脏灌注后取出嗅上皮，然后进行石蜡包埋。制作冠状切片进行HE染色以观察嗅上皮厚度变化，进行原位杂交（ISH）以研究上皮中嗅觉受体基因（Olr287、Olr226、Olr1493和Olr1654）的表达情况，同时评估嗅上皮再生过程中所选受体表达水平和位置的变化。

结果

HE染色显示，术后5天嗅上皮细胞数量和厚度明显减少，术后2周或4周逐渐增加。恢复6周后，上皮厚度可达到对照水平。ISH细胞染色模式显示沿前后（AP）和背腹（DV）轴有特定的空间分布。有证据表明，气味受体在正常或神经切断后恢复的上皮中呈连续且多个重叠带分布，而非传统认为的三或四个区域。数据还表明，由气味受体表达确定和定义的感觉神经元类型分布在术后6周恢复到正常或接近正常水平。同样，嗅神经切断术后5天，神经切断后恢复的标本中探针标记的神经元数量明显减少。手术侧Olr1493的反应性细胞（x(-) +/- s）为（53.9 +/- 19.9），未手术侧为（419.0 +/- 21.2），两者之间有统计学意义（t = 63.960，P < 0.01）。随着上皮再生，反应性细胞逐渐增加，6周后与正常侧几乎相等，无明显差异（t = 2.600，P > 0.05），手术侧和未手术侧的绝对阳性细胞分别为（417.8 +/- 32.4）和（445.3 +/- 10.0）。