两种亨廷顿蛋白相关蛋白1异构体—HAP1A和HAP1B在大鼠脊髓灰质内的分布特征  

DISTRIBUTIVE CHARACTER OF 2 ISOFORMS OF HUNTINGTIN-ASSOCIATED PROTEIN 1,HAP1A AND HAP1B,IN THE GRAY MATTER OF THE RAT SPINAL CORD

作　　者：韩金红[1,2] 李和[2] 

机构地区：[1]新乡医学院组织学与胚胎学教研室,河南453003 [2]华中科技大学同济医学院解剖学系组织学与胚胎学室,武汉430030

出　　处：《中国组织化学与细胞化学杂志》2010年第3期211-215,共5页Chinese Journal of Histochemistry and Cytochemistry

基　　金：国家杰出青年科学基金资助项目(30225024)

摘　　要：目的明确2种亨廷顿蛋白相关蛋白1(huntingtin-associated protein 1,HAP1)异构体—HAP1A和HAP1B在大鼠脊髓灰质内的分布特征。方法提取重组表达的谷胱甘肽S-转移酶(GST)-HAP1AC末端融合蛋白和GST-HAP1BC末端融合蛋白,免疫兔和豚鼠制备分别抗HAP1A和HAP1B特异性多克隆抗体,并采用免疫印迹技术对其特异性和效价进行鉴定和检测;用免疫组织化学技术检测HAP1A和HAP1B在大鼠脊髓灰质内的分布和定位特征。结果成功制备了分别特异性识别HAP1A和HAP1B的高效价多克隆抗体,应用这些抗体进行的免疫组织化学ABC法检测显示,HAP1A与HAP1B在大鼠脊髓灰质内的分布区域相似,二者均在大鼠脊髓灰质RexedⅠ-Ⅹ层表达,在RexedⅠ、Ⅱ层表达最强,中央管周围灰质(RexedX层)其次,在RexedⅢ-Ⅵ层表达水平较低,在脊髓前角(RexedⅦ-Ⅸ层)只有极微弱的或无阳性表达。HAP1A免疫反应产物主要定位在Stigmoid小体,在神经元胞质和近端突起内只有极少的弥散反应产物;HAP1B免疫反应性较强,其免疫反应产物弥散、均匀分布在神经元核周质和近端突起内,也定位在Stigmoid小体上。免疫荧光双重标记显示,约有80%的Stigmoid小体既表达HAP1A也表达HAP1B,其余的Stigmoid小体仅表达HAP1A。结论 HAP1A和HAP1B蛋白在大鼠脊髓灰质内具有相同的区域分布模式,但在神经元内的定位具有明显区别,提示二者可能具有不同的功能。Objective To reveal the distributive features of 2 isoforms of huntingtin-associated protein 1, HAP1A and HAP1B, in the gray matter of the rat spinal cord.Methods 2 recombinant proteins, the fusion protein of GST with C terminal of HAP1A and that of HAP1B,were produced and used as antigens to raise polyclonal antibodies specific for HAP1A and HAP1B, respectively, in rabbits and guinea pigs. The specificity and titers were identified by using immunoblotting. The distributive features of HAP1A and HAP1B in the gray matter of the rat spinal cord were detected by immunochemistry.Results Polyclonal antibodies specific for HAP1A and HAP1B, respectively, with high titers were successfully raised. Immunohistochemical ABC method using these antibodies detected that, in the gray matter of the rat spinal cord, the distributive regions of HAP1A were similar to those of HAP1B. Both HAP1A and HAP1B were expressed through Rexed layer I to X with the highst level in the Rexed I and II, second highest level in the gray matter around central canal （Rexed layer X）, lower level in the Rexed III to VI,and very low level or no expression in the ventral horn （Rexed layer VII to IX）. In the neurons, HAP1A-immunoreactive products were mainly localized in the Stigmoid bodies but very few in the perikaryon and proximal processes. The HAP1B-immunoreactivity was very strong. The immunoreactive products were diffusely and evenly distributed in the perikaryon and proximal processes. Some Stigmoid bodies were also positive for HAP1B immunoreactivity. Immunofluorescent double labelling showed that approximately 80% of Stigmoid bodies expressed both HAP1A and HAP1B,and the remaining expressed HAP1A only. Conclusion HAP1A has the same regional distribution pattern as HAP1B does in the gray matter of the spinal cord, but in the neurons, they have different localization, suggesting that HAP1A might have different functions from HAP1B.

关 键 词：亨廷顿蛋白相关蛋白1 大鼠 脊髓 免疫组织化学 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]