当归补血汤对耳蜗干细胞鼓阶内移植治疗药物性感音神经性耳聋大鼠增效作用研究

Effects of astragalus membranaceus and angelica sinensis on implantation of stem cells from cochlea for management of hearing loss

目的:观察当归补血汤对耳蜗干细胞移植治疗感音神经性耳聋大鼠增效作用。方法:感音神经性耳聋大鼠分为耳蜗干细胞移植组、当归补血汤组和空白对照组3组。体外培养耳蜗干细胞进行内耳移植,耳蜗干细胞移植组将耳蜗干细胞悬液移植入内耳耳蜗结构,当归补血汤组将含有当归补血汤的耳蜗干细胞悬液移植入内耳耳蜗结构,空白对照组移植生理盐水。分别于术后1、3、6和12个月采用听觉脑干诱发电位方法检测模型大鼠的听力恢复情况,处死大鼠,通过免疫荧光染色方法观察移植的耳蜗干细胞在内耳存活、分化和迁移情况。结果:从E14大鼠耳蜗分离的组织,经原代和传代培养后,可获得大量未分化悬浮生长的Nestin阳性细胞团,Brdu表达阳性,说明是具有自我更新和增殖潜能的干细胞。免疫荧光检测发现在耳蜗干细胞移植组、当归补血汤组在移植针道两侧可见移植的干细胞,有些还迁移到基底膜区。存活的细胞部分呈My-osinⅦA阳性染色。与耳蜗干细胞移植组比较,当归补血汤组MyosinⅦA阳性染色细胞数量较多,差异有统计学意义(P<0.05)。听觉脑干诱发电位结果显示当归补血汤组的感音神经性耳聋模型大鼠听力恢复情况较耳蜗干细胞移植组和对照组好,差异有统计学意义(P<0.05)。结论:经体外培养的耳蜗干细胞移植入内耳鼓阶后能够存活和迁移,并分化为内耳毛细胞;当归补血汤可以提高耳蜗干细胞分化为内耳毛细胞的比例,并促进感音神经性耳聋模型大鼠听力恢复。

Object： To investigate the effect of astragalus membranaceus and angelica sinensis on implantation of stem cells from cochlea for management of hearing loss.Methods： The hearing loss model rats were divided into cochlea stem cells group,astragalus membranaceus and angelica sinensis group,control group.Cochlea stem cells were isolated and cultured by using serum free medium.Cochlea stem cells were grafted into the rats＇ cochlea regions in cochlea stem cells group,both cochlea stem cells and astragalus membranaceus and angelica sinensis were grafted into the rats＇s cochlea regions in astragalus membranaceus and angelica sinensis group,and saline were grafted into the rats＇s cochlea regions in the control group.The hearing recovering of the hearing loss model rats were measured by brain stem auditory evoked protential on 1,3,6 and 12 months after implantation.Immunofluorescence was used to detect the results of stem cells survival,differentiation and migration.Results： The dissociated cells from the newborn rat organ of corti developed into much of indiffrentiated otopheres of nestin（＋） cells when plated on a non-adherent substratum.Within cultured otospheres,Brdu（＋） cells were rapidly risen to newly formed otopheres.Myosin ⅦA（＋） hair cells were detected by immunofluorescence on both side of implantation area in cochlea stem cells group and astragalus membranaceus and ngelica sinensis group,some of Myosin ⅦA（＋） hair cells migrated to basal membrane area.The number of Myosin ⅦA（＋） hair cells was much more in astragalus membranaceus and angelica sinensis group than in other groups（P0.05）.The hearing recovering of the hearing loss was better in astragalus membranaceus and angelica sinensis group than in other groups（P0.05）.Conclusion： Cochlea stem cells could survive,differentiate and migrate after implantation into the hearing loss model rats.Astragalus membranaceus and angelica sinensis could increase the number of Myosin ⅦA（＋） hair cells and accelerate hearing recovering of the hearing loss model rats.