Chai Liang, Gao Yang, Gu Zhi-yan, Ni Dao-feng
Department of Otorhinolaryngology, Peking Union Medical College Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing 100730, China.
Zhonghua Er Bi Yan Hou Tou Jing Wai Ke Za Zhi. 2005 Aug;40(8):561-5.
To investigate the protein and mRNA expression patterns of apoptosis-related genes, together with evidence of apoptosis, in relation to experimental autoimmune inner ear disease (AIED).
Male C57BL/6 mice at 4 weeks age (n = 80) were randomly assigned to one of the five group (n = 16). The inbred mice were given a single subcutaneous injection of diluted solution of pertussis and an emulsion containing equal parts of complete Freund adjuvant (CFA) and inner ear antigens (IEAg) extracted form guinea pig. The animals were sacrificed for inner ear examination at a defined time after the immunization (7, 14, 21 or 28 days). An autoimmune inner ear diseases model was established. Apoptosis was detected by terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (d-UTP) nick end-laying (TUNEL) method. Using immunohistochemical techniques and reverse transcriptase polymerase chain reaction to clarify the profile of Fas, FasL, and bcl-2.
Under normal conditions, no TUNEL-positive cell was observed in the cochlea except for a few positive cells in the supporting cells of Corti's organ and macula sacculi. Inner ear antigens administration induced TUNEL-positive reactions in a wide variety of cells such as inner hair cells, supporting cells, stria vascularis and spiral ligament fibrocytes. No positive staining was evident in outer hair cells, spiral ganglion cells and Scarpa's ganglion cells during the whole period. Fas proteins were expressed in a wide range of cells in inner ear. The levels of Fas mRNA were no significant differences between normal and AIED mice. FasL and bcl-2 proteins could be detected in spiral ganglion cells and Scarpa's ganglion cells both in normal and AIED mice. FasL positive cells increased in number in inner ear of AIED mice. bcl-2 positive cells were not detectable in inner hair cells, stria vascularis and spiral ligament both in normal and AIED mice. The mRNA of three kinds of apoptosis-related genes was detectable in the normal and AIED mice. FasL mRNA was expressed at low levels in normal, being maximal at 14 d post inoculation and decreased gradually to steady levels by 2 weeks. The levels of bcl-2 mRNA increased significantly during the period of AIED.
Apoptosis mediated by Fas/FasL signal system may play a role in the initiation and maintenance of AIED. bcl-2 has a crucial role in the regulation of the process of apoptosis in the inner ear of AIED mice.
研究凋亡相关基因的蛋白质和mRNA表达模式以及凋亡证据,探讨其与实验性自身免疫性内耳疾病(AIED)的关系。
将80只4周龄雄性C57BL/6小鼠随机分为5组(每组16只)。给近交系小鼠皮下注射百日咳稀释液以及含有等量完全弗氏佐剂(CFA)和从豚鼠提取的内耳抗原(IEAg)的乳剂。在免疫后特定时间(7、14、21或28天)处死动物进行内耳检查,建立自身免疫性内耳疾病模型。采用末端脱氧核苷酸转移酶(TdT)介导的脱氧尿苷三磷酸(d-UTP)缺口末端标记(TUNEL)法检测凋亡。运用免疫组织化学技术和逆转录聚合酶链反应来明确Fas、FasL和bcl-2的表达情况。
正常情况下,除了柯蒂器支持细胞和球囊斑中有少数阳性细胞外,耳蜗中未观察到TUNEL阳性细胞。给予内耳抗原后,在内毛细胞、支持细胞、血管纹和螺旋韧带纤维细胞等多种细胞中诱导出TUNEL阳性反应。在整个观察期间,外毛细胞、螺旋神经节细胞和斯卡帕神经节细胞均无明显阳性染色。Fas蛋白在内耳多种细胞中表达。正常小鼠和AIED小鼠的Fas mRNA水平无显著差异。正常小鼠和AIED小鼠的螺旋神经节细胞和斯卡帕神经节细胞中均可检测到FasL和bcl-2蛋白。AIED小鼠内耳中FasL阳性细胞数量增加。正常小鼠和AIED小鼠的内毛细胞、血管纹和螺旋韧带中均未检测到bcl-2阳性细胞。正常小鼠和AIED小鼠中均可检测到三种凋亡相关基因的mRNA。FasL mRNA在正常时低水平表达,接种后14天达到最高,2周后逐渐降至稳定水平。AIED期间bcl-2 mRNA水平显著升高。
Fas/FasL信号系统介导的凋亡可能在AIED的发生和维持中起作用。bcl-2在AIED小鼠内耳凋亡过程的调节中起关键作用。