Stacey D J, McLean W G
Department of Pharmacology and Therapeutics, University of Liverpool, L69 3BX, Liverpool, UK.
Brain Res. 2000 Jul 21;871(2):319-32. doi: 10.1016/s0006-8993(00)02488-4.
In birds, spontaneous recovery of the hair cells of the inner ear can occur after damage induced by aminoglycoside antibiotics. The factors that influence this recovery and the process of hair cell regeneration itself have until recently been investigated largely by morphological and histological methods. The aim of this work was to use a molecular biological approach to the analysis of hair cell regeneration by measuring the changes that occur in expression of mRNA for hair cell-specific cytoskeletal proteins fimbrin and class III beta-tubulin, along with that for beta-actin, in the utricle of chicks after hair cell damage both in vitro and in vivo. Utricles were removed from 1-day-old chicks and incubated with the aminoglycoside antibiotics gentamicin or neomycin (both 1 mM), or chicks were injected intraperitoneally with 100 mg/kg gentamicin or neomycin for 4 consecutive days. At the end of the treatment periods, total RNA was extracted from single utricles, reverse transcribed to cDNA and the cDNA amplified by PCR with primers for beta-actin, fimbrin and class III beta-tubulin. Co-amplification allowed quantitative comparison of mRNA between fimbrin, or class III beta-tubulin and beta-actin from the same utricle. Both aminoglycosides, either after 48 h in vitro or immediately after treatment in vivo, caused a significant decrease in the expression of fimbrin mRNA and class III beta-tubulin mRNA, relative to beta-actin mRNA, which itself increased. Light and electron microscopy confirmed that this corresponded to loss of, and damage to, hair cells. The relative expression of fimbrin and class III beta-tubulin mRNAs was partly restored almost to control levels 4 days after cessation of treatment in vivo and fully normalised by 4 weeks, by which time hair cells appeared normal. However, their relative expression remained depressed 4 days after removal of antibiotic in vitro. The iron chelator desferrioxamine (100 microM) in vitro prevented the aminoglycoside-induced reduction in relative expression of mRNA for both fimbrin and class III beta-tubulin. Neither insulin (5 microM) nor a combination of dibutyryl cyclic AMP (5 mM) and the phosphodiesterase inhibitor IBMX (0.5 mM) prevented the decrease in relative expression of the mRNAs for the hair cell-specific proteins, but both treatments allowed their partial recovery in vitro during the 4-day-period after removal of aminoglycoside. It is concluded that the cells of the sensory epithelium of the chick utricle subjected to aminoglycoside-induced damage undergo a process in which mRNA expression is switched away from the production of functional proteins and towards proteins necessary for structural re-organisation. The restoration of mRNA expression to a normal pattern is promoted by the growth factor insulin and by cyclic AMP.
在鸟类中，内耳毛细胞在受到氨基糖苷类抗生素诱导的损伤后能够自发恢复。直到最近，影响这种恢复的因素以及毛细胞再生过程本身主要是通过形态学和组织学方法进行研究的。这项工作的目的是采用分子生物学方法，通过测量雏鸡椭圆囊在体内外毛细胞损伤后，毛细胞特异性细胞骨架蛋白丝束蛋白和Ⅲ类β-微管蛋白以及β-肌动蛋白的mRNA表达变化，来分析毛细胞再生。从1日龄雏鸡中取出椭圆囊，用氨基糖苷类抗生素庆大霉素或新霉素（均为1 mM）进行孵育，或者雏鸡连续4天腹腔注射100 mg/kg庆大霉素或新霉素。在治疗期结束时，从单个椭圆囊中提取总RNA，逆转录为cDNA，并用针对β-肌动蛋白、丝束蛋白和Ⅲ类β-微管蛋白的引物通过PCR扩增cDNA。共扩增允许对来自同一椭圆囊的丝束蛋白或Ⅲ类β-微管蛋白与β-肌动蛋白之间的mRNA进行定量比较。两种氨基糖苷类抗生素，无论是在体外处理48小时后还是在体内处理后立即观察，相对于自身增加的β-肌动蛋白mRNA，都会导致丝束蛋白mRNA和Ⅲ类β-微管蛋白mRNA的表达显著下降。光学和电子显微镜证实，这与毛细胞的丧失和损伤相对应。在体内停止治疗4天后，丝束蛋白和Ⅲ类β-微管蛋白mRNA的相对表达部分恢复到几乎对照水平，并在4周时完全恢复正常，此时毛细胞看起来正常。然而，在体外去除抗生素4天后，它们的相对表达仍然受到抑制。体外铁螯合剂去铁胺（100 microM）可防止氨基糖苷类抗生素诱导的丝束蛋白和Ⅲ类β-微管蛋白mRNA相对表达的降低。胰岛素（5 microM）以及二丁酰环磷腺苷（5 mM）与磷酸二酯酶抑制剂异丁基甲基黄嘌呤（0.5 mM）的组合均不能防止毛细胞特异性蛋白mRNA相对表达的降低，但两种处理都能使它们在体外去除氨基糖苷类抗生素后的4天内部分恢复。结论是，受到氨基糖苷类抗生素诱导损伤的雏鸡椭圆囊感觉上皮细胞经历了一个过程，其中mRNA表达从功能性蛋白的产生转向结构重组所需蛋白的产生。生长因子胰岛素和环磷腺苷促进mRNA表达恢复到正常模式。
