抗分泌因子与水通道蛋白1和2在大鼠内耳中的表达及其相互作用

目的研究抗分泌因子（antisecretory factor，AF）与水通道蛋白1、2（aquaporin-1，2，AQP1，2）在大鼠内耳中的表达及其相互作用。方法选取6只健康雄性SD大鼠，采用Envision二步法免疫组化技术，观察AF、AQP1和AQP2在大鼠内耳中的表达情况；选取20只健康雄性SD大鼠，分别取其前庭及耳蜗组织，运用免疫共沉淀结合蛋白质印记方法，用抗AQP1的单克隆抗体和抗AQP2多克隆抗体分别特异性地沉淀前庭和耳蜗组织中的蛋白抗原，用抗AF的特异性抗体检测沉淀物。结果AF在内耳分布广泛，耳蜗血管纹边缘细胞、螺旋韧带Ⅰ-Ⅴ型纤维细胞、前庭膜、基底膜、壶腹嵴等部位呈轻中度阳性反应，圆窗膜呈中强阳性反应，耳蜗螺旋神经节及前庭、耳蜗神经纤维均有阳性分布；AQP1主要分布于血管纹的中间细胞、螺旋韧带Ⅲ型纤维细胞、基底膜以及圆窗膜，染色强度为中重度；AQP2主要表达于螺旋韧带Ⅱ型、Ⅳ及Ⅴ型纤维细胞，呈中重度阳性染色反应，圆窗膜也有轻度表达。以AF特异性抗体分别检测AQP1及AQP2特异性抗体沉淀物中有清晰的阳性条带，相对分子质量约60000，与AF的相对分子质量吻合，而未加AQP1及AQP2特异性抗体的对照实验中无反应条带出现。结论AF、AQP1及AQP2在内耳组织中的分布有一定的规律，多位于与内淋巴关系密切的部位，提示三种蛋白可能参与内淋巴中水的调节。AF的分布区与AQP1及AQP2均有重叠，AQP1、AQP2与AF之间均存在相互结合作用。

Expression of antisecretory factor and aquaporin 1,2 in the rat inner ear and their interaction

OBJECTIVE: To study the interaction among aquaporin1 (AQP), aquaporin2 (AQP2) and antisecretory factor( AF) , and their expression in the rat inner ear for furthur understanding of Meniere' s disease. METHODS: Inner ear tissue section of six healthy male Speague-Dawley rats was performed and Envision immunochemical staining was applied to detect the expression of AF, AQP1 and AQP2 in the rat inner ear. Vestibular and cochlear tissues of twenty healthy male Speague-Dawley rats were dissected. Coimmunoprecipitation and Western Blot were used to specifically immunoprecipitate AF protein in the vestibular and cochlear tissues with monoclonal antibodies against AQP1 and polyclona antibodies antibodies against AQP2 to detect the above precipitate with specific antibodies against AF. RESULTS: (1) AF was widely distributed in the inner ear, such as marginal cells of stria vascularis , five classes of spiral ligment fibrocyte , Reissner's membrane, basilar membrane, ampullar crest and so on with mild or moderate staining. In addition, round membrane was moderately or markedly stained. Positive immunostaining was found in the cochlear spiral ganglion, vestibular nerve and cochlear nerve. AQP1 was distributed in the intermediate cells in stria vascularis, type III fibrocyte of spiral ligament, basilar membrane and round membrane with moderate to marked degree of immunostaining intensity. AQP2 was mainly localized to the type II, IV, and V fibrocyte of spiral ligament, with moderate to marked degree of immunostaining intensity, round membrane was weakly stained. (2) No band was observed in the control and a single immunoreactive band of 60 000 was observed, which was equal to the molecular mass of AF. CONCLUSIONS: (1) AF, AQP1 and AQP2 have its individual specific localization in the rat inner ear, which was close to the parts of endolymph, so regulating water of the endolymph may be possible. (2) The range of localization of AF overlapped the distribution of AQP1 and AQP2. The results showed the existence of AF protein in the immunoprecipitate using co-immunoprecipitation combined with Western Blot. It suggested that the interreaction between AQP1, AQP2 and AF might be possible.