内耳免疫反应中细胞凋亡及Caspase-3动态表达的研究

目的:研究内耳免疫反应过程中是否存在细胞凋亡,以及细胞凋亡是否与Caspase3信号转导有关。方法:选用雌性白色豚鼠45只,随机分为实验组29只,对照组16只,以钥孔虫戚血蓝蛋白(KLH)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(PBS),分别在内耳免疫术后1、3、5、7d和14d后处死动物,于免疫前及处死前行双侧听性脑干反应(ABR)测试。取内耳免疫侧耳蜗做石蜡切片。通过DNA末端转移酶介导的缺口末端标记法(TUNEL)检测内耳凋亡细胞,免疫组织化学检测内耳Caspase3的表达。结果:实验组豚鼠内耳免疫前、后反应阈比较有统计学意义(P<0.05或P<0.01);对照组给药前、后比较反应阈无明显变化。实验组豚鼠内耳均存在TUNEL染色阳性细胞,并且TUNEL染色阳性细胞具有凋亡细胞的典型形态学特征,而对照组仅在支持细胞、血管纹和螺旋神经节细胞中发现极少数TUNEL染色阳性细胞。Caspase3免疫组织化学染色实验组内耳从内耳免疫后5d开始出现阳性表达,而对照组表达阴性。结论:内耳免疫反应可以诱导细胞凋亡的发生;内耳免疫反应诱导细胞凋亡的发生中有Caspase3的激活。

Apoptosis and dynamic Caspase-3 expression during the immune response of inner ear

OBJECTIVE: To investigate whether apoptosis is invovled in the pathogenesis of the immune response of inner ear, and relation between signal transportation of Caspase-3 and apoptosis. METHOD: Forty-five healthy, female white guinea pigs were employed in this study. 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. All animals were sacrificed respectively at 1st, 3rd, 5th, 7th and 14th day after inner ear vaccination. The auditory brainstem response (ABR) was measured before the KLH injection to the inner ear and just before sacrificing the animal. Paraffin sections of cochleas from animals were stained using a TUNEL assay to identify inner ear cells undergoing apoptosis,and immunohistochemistry method was used to detect the expression of Caspase-3 in the inner ears. RESULT: After immunization, the ABR thresholds of the KLH-challenged ears were significantly elevated by comparing with the preimmunization (P < 0.05), but after injection, there was no significant difference in the ABR thresholds of the PBS-injected ears compared with preinjection. TUNEL-positive cells were found in the KLH immunized inner ears but no TUNEL-positive cells were observed in the control inner ears except for a few positive cells in the supporting cells, the stria vascularis cells and the spiral ganglion cells. The positive cells are the outer hair cells in Cortis organ, and the marginal cells in the stria vascularis and the neurons in the spiral ganglion. And, under morphological analysis by light microscope, these cells have the features characteristic of apoptosis. Apparent Caspase-3 immunoreactivity expression could be detected in Corti's organ, the lateral wall and the neurons of the spiral ganglion in the KLH-immunized inner ears from 5th day after inner ear vaccination, but no cells staining positive for Caspase-3 were found in the control inner ears. CONCLUSION: Apoptosis is induced by the immune response of inner ear; Caspase-3 cascade has important effect on the course of apoptosis.