Analysis of Connexin 32 Antigen Expression in Experimentally Degenerative Peripheral Nerve Using A Monoclonal Antibody.

连接蛋白32（Cx 32）在正常周围神经中被识别，我们已经报道了抗Cx 32的单克隆抗体，命名为HAM 8。近年来，有报道称，在人的周围神经退行性变中，Cx 32不表达，如腓骨肌萎缩症。本课题的目的是利用大鼠周围神经退行性变模型和人类疾病来阐明Cx 32在周围神经退行性变中的存在或消失。用间接免疫荧光法检测大鼠坐骨神经冰冻切片中Cx 32的存在。切片中存在HAM 8阳性或阴性区域。然而，我们无法详细显示Cx 32在神经中的显微定位。然后，我们对正常大鼠离体神经纤维的Cx 32定位进行了一些改进，如固定液、固定时间、Triton-X浓度和时间的变化。神经纤维在3%福尔马林溶液中浸泡20 min或1 h后，再用0.5%、1%和2 % Triton-X溶液处理10 min或1 h，Cx 32信号较其他条件处理的神经纤维标本清晰。定位于除Rvier淋巴结外的沿着髓鞘。此外，我们还用激光共聚焦显微镜观察了Cx 32的定位，但不能确定Cx 32是否位于包裹轴突的髓鞘之间。我们建立了大鼠神经退行性变模型的制作方法，如（1）放射大鼠神经，（2）GVH大鼠神经和（3）Waller变性神经。它们和病人的人体标本保存在深冻或液氮中。

Connexin (Cx) 32 is recognized in normal peripheral nerve, and we have already reported anti-Cx32 monoclonal antibody which was named HAM8. Recently, it was reported that Cx32 was not epxressed in human degenerative peripheral nerve from the patients suffering from such as Charcot-Marie-Tooth disease. The purpose of this project was to clarify the existence or disapperance of Cx32 in degenerative peripheral nerve using a rat model and human disease.In the beginning, we examined the expression of Cx32 in normal rat peripheral nerve. We could show the existence of Cx32 in the frozen section of rat sciatic nerve using indirect immunofluorescence method. There were HAM8 positive or negative territories in the section. However, we could not show in detail the microscopic localization of Cx32 in the nerve. Then, we exmined the localization of Cx32 in the dissociated nerve fibers from normal rat with some modifications ; changes in fixative solution, duration period for fixing and concentration and duration peiriod of Triton-X.It was not being completely succeeded to visualize the Cx32 localization in normal rat peripheral nerve until now. When nerve fibers were immersed in 3 % formalin solution for 20 min or 1 hr then they were treated with 0.5,1 or 2 % Triton-X solution for 10 min or 1 hr, the signal from Cx32 was clearer than the nerve fiber preparations treated with other conditions examined. The localization was recognized along the myelin sheath besides Rvier's nodes. Furthermore, we examined the localization using con-focal LASER microscopy, but could not determine whether Cx32 was located between each myelin sheet wrapping the axons.We have established the methods to make the degenerative nerve model in rat, such as (1) nerve from irradiated rats, (2) nerve from GVH rats and (3) nerve from Waller degeneration. They and human specimen from patients were kept in deep freezer or liquid nitrogen.