缺氧对体外培养大鼠耳蜗毛细胞的损伤作用

目的: 建立耳蜗器官体外培养模型, 观察缺氧对体外培养耳蜗内毛细胞 （IHC）及外毛细胞（OHC）的影响。方法: 分离生后3 d Wistar大鼠耳蜗基底膜, 平铺在含有DMEM培养液的培养基内 （每盘6条基底膜）,2盘为1组,随机分为8组,放置在37℃、5％CO₂培养箱培养，其中7组作为实验组,按不同时间段进行缺氧（37℃、90％N₂、5％CO₂、5％O₂） 培养；1组作为对照组放置在37℃、5％CO₂培养箱。采用Phalloidin 荧光标记观察耳蜗中IHC及OHC形态变化并计数单位面积（24 mm×36 mm）细胞数即细胞密度。结果：在缺氧早期（0.5 h）IHC形态及密度无明显变化;1 h细胞轻度肿胀,体积增大,单位面积细胞数减少,细胞密度与对照组比较差异无显著性 （P>0.05）;2 h时 IHC散在缺失, 细胞密度与对照组比较差异有显著性（P<0.01）; 6 h时 IHC缺失增多,并可见毛细胞坏死后遗留的“空神经杯”现象,细胞密度与对照组比差异有显著性（P<0.01）; 随缺氧时间延长, IHC缺失逐渐加重。OHC在缺氧早期（0.5 h、1 h）形态学改变不明显, 2 h偶见细胞缺失, 随缺氧时间延长, 6 h后出现不同程度的排列拥挤、紊乱及缺失, 单位面积细胞数目减少, 细胞密度与对照组比较差异有显著性（P<0.01）, 以48 h最严重。结论: 缺氧造成体外培养耳蜗毛细胞缺失, 以IHC为重。

Effect of hypoxia on cultural hair cells of rat cochlea in vitro

Objective To establish a practical model for Wistar rat cochlea organ cultivated in vitro and observe the effect of hypoxia on the inner hair cells(IHC) and outer hair cells(OHC).Methods The cochlear basal membrane from 3-day-old Wistar rats was used.The cochlear basal membrane cells were divided into 8 groups.Among them 7 groups were used as experimental groups.The cells in experimental groups were put in incubator(37℃,90%N2,5%CO2,5%O2)to cultivate with hypoxia for different time.The other group was used as control,the cells were cultivated in incubator(37℃,5%CO2).The cultures were phalloidin-labeled and the number of IHC / OHC were counted in unit area(24 mm×36 mm) and the morphological changes of IHC and OHC were observed under microscope.Results No significant change of IHC was found under hypoxia for 0.5 h.IHC under hypoxia for 1 h appeared light swelling and cell density decreased,but the cell density in unit area had no remarkable difference compared with control(P>0.05).IHC loss was found under hypoxia for 2 h to 48 h in a time dependent manner,the cell density in unit area had remarkable difference compared with control(P<0.01).The phenomena of empty nerve cup was found under hypoxia for 6 h.The OHC loss and array disorders appeared under hypoxia for 6 h and severe damage under hypoxia for 48 h,the cell density in unit area had remarkable difference compared with control(p<0.01).Conclusion Hypoxia could cause the hair cell loss in vitro.Moreover IHC is more susceptible to hypoxia than OHC.