缺氧对体外培养大鼠耳蜗毛细胞的损伤作用

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

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3d 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。

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）. Oonclusion Hypoxia could cause the hair cell loss in vitro. Moreover IHC is more susceptible to hypoxia than OHC.