耳蜗血管纹组织块的缘细胞培养

Culture of marginal cells from guinea pig cochlear stria vascularis explants

目的 :建立豚鼠耳蜗血管纹 (SV)组织块缘细胞 (MCs)的培养方法 ,为进一步研究药物耳毒性及其作用机制奠定基础。方法 :2 6只豚鼠按SV培养时间随机分成 4组 :2 4h组 (n =8) ;72h组 (n =8) ;>72h组 (n =8) ;对照组 (新鲜SV固定组 ,n =2 )。显微解剖数段连同螺旋韧带的SV组织块 ,置于 5 %CO2 / 95 %空气的二氧化碳恒温 (37℃ )培养箱中进行培养 ,分别进行形态学和组织学观察。结果 :培养 2 4hSV组织块保持良好活性 ,其组织学结构与新鲜固定的SV结构无明显差异 ;培养 72hSV组织块与新鲜固定的SV在组织学结构方面有显著性差异 ,不能观察到正常的SV结构 ,组织结构松散 ,缘细胞从组织块离心性生长出来 ;从SV组织块培养出的缘细胞能在培养皿内存活 13d。结论 :采用组织块培养技术 ,成功地建立了豚鼠耳蜗SV组织块的缘细胞培养方法 ;培养 2 4h的SV组织块光镜下保持了良好活性和正常组织学结构 ,可用来进一步研究药物耳毒性及其作用机制。

Objective:To provide the prerequisite for further investigation of drugs ototoxicity and the probable mechanisms,explant culture technique of guinea pig cochlear strial marginal cells were established.Method: 26 healthy pigmented guinea pig were randomly divided into four groups according to the period of stria vascularis cultivated: 24 hours group (n=8); 72 hours group (n=8); more than 72 hours group (n=8); control group (fresh stria vascularis fixed group, n=2). Several explants of stria vascularis and spiral ligament obtained by mechanical dissociation were cultivated and kept at 37℃with maximal humidity in 5% CO 2 /95% air. The following culture media were used: E MEM with hepes buffer (20 mmol/L), fetal calf serum (10%). Result: The normal activity of stria vascularis explants cultivated for 24 hours may maintain. There were no significant change in the structure features of stria vascularis between 24 hour group and control group. There were significant difference in the structure features between 72 hour group and control group.The stria vascularis of 72 hour group cannot be observed normal stria vascularis structure,the stria vascularis structure was loose,the marginal cells reached the border of the explant and they proliferated outside the explant.The marginal cells cultivated from the stria vascularis explant may be cultivated in cell culture dishs for 13 days.Conclusion: Our present results suggested that explant culture technique of cochlear strial marginal cells of guinea pigs has been successfully established.The stria vascularis cultivated for 24 hours which be maintained normal activity and structure features may be used for further investigation of drugs ototoxicity and the probable mechanisms.