内耳免疫反应中细胞凋亡的研究

目的探讨内耳免疫反应过程是否引起细胞凋亡以及Fas和FasL、Bcl-2和Bax的表达情况。方法选用雌性白色豚鼠16只，随机分为实验组和对照组各8只，以钥孔蛾血蓝蛋白全身免疫后，实验组以相同抗原进行内耳免疫，对照组内耳注射等量的磷酸盐缓冲生理盐水，在内耳免疫7d后处死动物，取内耳免疫侧耳蜗做石蜡切片。通过电镜和脱氧核糖核苷酸末端转移酶介导的缺口末端标记技术(terminal-deoxynucleotidyl transferase mediated nick end labeling，TUNEL)检测内耳凋亡细胞，免疫组化检测内耳Fas和FasL以及Bcl-2和Bax的表达。结果透射电镜观察发现实验组术后7d内耳外毛细胞、血管纹细胞及螺旋神经节细胞都出现了凋亡细胞的特征性改变，而对照组未发现具有上述特征的细胞。实验组内耳Corti器毛细胞，血管纹的缘细胞和螺旋神经节细胞存在TUNEL染色阳性细胞，TUNEL染色阳性细胞具有凋亡细胞的典型形态学特征，对照组内耳的任何结构中都没发现TUNEL染色阳性细胞。免疫组化染色实验组Corti器、螺旋神经节细胞、血管纹和螺旋韧带Fas和FasL蛋白表达阳性，而对照组只有螺旋神经节细胞和血管纹有较弱的Fas蛋白表达，FasL蛋白表达阴性。实验组Corti器、螺旋神经节细胞、侧壁Bcl-2蛋白表达阴性，对照组的Corti器、侧壁和螺旋神经节细胞Bcl-2蛋白表达阳性。实验组Corti器、侧壁和螺旋神经节细胞Bax蛋白表达阳性，对照组只有螺旋神经节细胞Bax蛋白表达弱阳性，Corti器、侧壁表达阴性。结论内耳免疫反应可诱导细胞凋亡发生，Fas-FasL是此过程的信号转导途径之一，Bcl-2和Bax蛋白在其中起了重要调节作用。

Apoptosis in the immune response of inner ear]

OBJECTIVE: To investigate whether apoptosis is one of the mechanism in the immune response of inner ear,and to detect the expression of Fas, FasL, Bcl-2 and Bax in the inner ears. METHODS: Sixteen healthy, female guinea pigs were employed in the experiment. Sensitized systematically with keyhole limpet hemocyanin (KLH), the KLH-immunized animals were inoculated with the same antigen, and the control animals were injected PBS through cochlea basal turn. The animals were sacrificed at 7 day after inner ear vaccination. Transmission electron microscopy was used to detect inner ear apoptotic cells, and paraffin sections of cochlea from animals were stained using a terminal-deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay to identify inner ear cells undergoing apoptosis. Immunohistochemistry method was used to detect the expression of Fas, FasL, Bcl-2 and Bax in the inner ears. RESULTS: The observation of electron microscopy had shown the features characteristic of apoptotic cells in the KLH-immunized inner ears but not in the control inner ears. TUNEL-positive cells were found in the KLH-immunized inner ears but not in the control inner ears. The positive cells were the hair cells in Corti's organ, and the marginal cells in the stria vascularis and the neurons in the spiral ganglion. Moreover under morphological analysis by light microscope, these cells had the features characteristic of apoptosis. High expression of Fas and FasL could be detected in Corti's organ, the stria vascularis, the spiral ligament and the neurons of the spiral ganglion in the KLH-immunized inner ears. A low expression of Fas could be detected in the stria vascularis and the neurons of the spiral ganglion in the control inner ears, but no cells staining positive for FasL were found in the control inner ears. No cells staining positive for Bcl-2 were found in the KLH-immunized animals but moderate expression of Bcl-2 could be detected in Corti's organ, the lateral wall and the neurons of the spiral ganglion in the control inner ears. High expression of Bax could be detected in Corti's organ, the lateral wall and the neurons of the spiral ganglion in the KLH-immunized inner ears. A low expression of Bax could be detected in the neurons of the spiral ganglion and no cells staining positive for Bax were found in Corti's organ, the lateral wall in the control inner ears. CONCLUSIONS: These findings suggest apoptosis is involved in the pathogenesis of the immune response of inner ear and Fas- FasL pathway is one of important signal transportation of the course and Bcl-2 and Bax have a critical role in the regulation of apoptotic cell death induced by the immune response of inner ear.