胆红素对离体小鼠耳蜗组织毒性作用的研究

Toxic Effects of Bilirubin on Mouse Cochlea

目的观察不同浓度胆红素对离体培养小鼠耳蜗组织的损伤效应。方法取新生5天的C57BL/6J小鼠,解剖获得耳蜗基底膜组织离体培养48小时,将耳蜗基底膜组织随机分为四组:对照组、25μmol/L胆红素组、50μmol/L胆红素组、100μmol/L胆红素组。实验组培养基按照分组浓度,加入相应胆红素溶液,对照组加入相应容积的生理盐水。所有标本继续培养24小时后终止,进行组织固定和免疫荧光染色,通过激光共聚焦显微镜观察,最后行统计学分析。结果不同浓度胆红素作用后,25μmol/L胆红素组中耳蜗神经纤维减少,50μmol/L胆红素组中神经纤维明显减少,100μmol/L胆红素组中神经纤维几乎完全消失,而各组内外毛细胞形态和数量无变化。各胆红素组的神经纤维数与对照组有统计学差异(P<0.05),且各胆红素组之间的神经纤维数有统计学差异(P<0.05)。结论胆红素对于离体培养的新生小鼠的耳蜗神经纤维的毒性作用呈浓度依赖性;在胆红素浓度不超过100μmol/L时,胆红素对于离体培养的新生小鼠的耳蜗组织的内毛细胞和外毛细胞不造成损害。

Objective To study effects of bilirubin on cultured mouse cochlear tissue at various concentrations.Method Cochlear basilar membranes containing spiral ganglions were gently isolated from P5 mice and used for experiments after 48 hours in culture.The basilar membranes were randomly divided into four groups to be exposed to bilirubin at 25μmol/L,50μmol/L or 100μmol/L in the culture media,or serve as controls(exposed to saline in the culture media).Specimens from each group were fixed and underwent immunofluorescence staining.The number and morphology of the nerve fibers and hair cells were examined under laser confocal microscope.Result The number of nerve fibers decreased as concentration of bilirubin increased and almost disappeared at 100μmol/L,although with no obvious change in hair cell morphology and quantity.Kruskal-Wallis test confirmed significant difference between the number of the nerve fibers in bilirubin exposed groups and that in the control group(P<0.05)and among the bilirubin exposed groups themselves(P<0.05).Conclusion The toxic effect of bilirubin on cultured newborn mice cochlear nerve fibers is concentration dependent.With concentration under 100μmol/L,bilirubin causes no damage to inner and outer hair cells in vitro.