新生大鼠耳蜗Corti器体外培养及其转基因表达

目的建立新生大鼠耳蜗Corti器体外培养模型及观察外源基因表达。方法采用出生后1~3天龄大鼠,分离出的耳蜗Corti器组织置入表面覆盖有胶原凝胶的培养皿中,在含有10%DMEM液体培养基中培养;加入携带报告基因的腺病毒悬液,观察外源基因的表达。结果耳蜗Corti器体外培养8小时后,培养组织生长良好,形态结构清晰可辨,在该实验条件下,Corti器形态结构可以维持5天以上,最长达7天,随着培养时间的延长,由于周围组织生长的蔓延,Corti器的结构分界不清楚。腺病毒加入Corti器后12小时即有基因表达,24小时达到高峰,表达时间可持续1周。结论新生大鼠离体内耳组织转基因培养法是内耳Corti器体外培养实验研究的一种可行方法。

Culture of the Organ of Corti of the Newborn Rat in Vitro and Transgene Expression

Objective To establish an model of the organ of Corti of the neonatal rat in vitro and to observe the expression of reported gene in the tissue.Methods Newborn rats were used in this study.Rats were sacrificed by decapitation and sterilized.The temporal bones were removed and the cochlear tissue was preserved with cold Hanks' balanced salt solution.Under stereomicroscope, the apex and the round window were opened, and the cochlear bony wall was removed.Then,the dissected explants were immediately put on culture dishs with the collagen layer filled with DMEM medium. Adenovirus suspension was slowly injected into the cochlear explants for transgene expression.Results The organ of Corti of the neonatal rat grew well 8 hours after the tissue cultivation.It maintained a bright and organized structure.The explants were maintained up to 5 days under the present experimental conditions.With time,the architecture of the organ was disturbed by the continuous growth and spread of the surrounding tissue.The cultures of the organ of Corti began to express reported gene 12 hours after transfection of virus. Gene expression reached high level after 24 hours, and continued for 1 week.Conclusion The model of culture of the organ of Corti in vitro has been established successfully and the reported gene expresses in the tissue.This provides a useful approach to studying the culture of organ of Corti in vitro.