骨髓神经组织定向干细胞移植治疗大鼠听神经损伤的实验研究

Experimental study of treatment of acoustic nerve injury in rats by bone marrow NTCSCs transplantation

目的观察骨髓神经组织定向干细胞(nerve tissue committed stem cells,NTCSCs)移植治疗大鼠听神经损伤的作用。方法将60只SD大鼠按数字随机法分为5组,每组各12只,A组为对照组,在平静饲养环境下培养,另外4组(B、C、D、E组)构建感音神经性耳聋动物模型。B组大鼠断头后取出听泡组织行HE常规染色,详细观察耳蜗螺旋神经节神经元(spiral ganglion neurons,SGNs)损伤和内耳毛细胞存活情况,确认造模成功。将绿色荧光蛋白(green fluore scent protein,EGFP)基因表达阳性的骨髓NTCSCs注射至C、D、E 3组大鼠左耳耳蜗蜗轴处,SD大鼠右耳及A组作为对照;采用同一方法注射相同体积的0.1%PBS溶液。HE染色观察耳蜗中轴切片,在荧光显微镜下观察转染EGFP基因的NTCSCS存活、分布部位及表达情况。结果 C组(移植后1周)内耳耳蜗切片上发现转染绿色荧光蛋白(green fluore scent protein,EGFP)基因的骨髓NTCSCs分散在鼓阶的腔隙内,D组(移植后2周)发现转染EGFP基因的骨髓NTCSCs位置靠近基底膜和柯蒂氏器部位,E组(移植后4周)发现转染EGFP基因的骨髓NTCSCs球团和多个在一起的细胞聚集,并且位置靠近基底膜和柯蒂氏器部位,呈现良好的分布,类似有向基底膜和柯蒂氏器迁移行为。随着骨髓NTCSCs移植时间的延长,Nestin(+)细胞数量明显增多(P<0.05),而MyosinⅦa(+)细胞数量无明显变化(P>0.05)。结论骨髓NTCSCs移植大鼠耳蜗后可在一定程度上修复损伤听神经。

ObjectiveTo observe the effect of bone marrow transplantation with nerve tissue committed stem cell （NTCSCs） on acoustic nerve injury in rats.Methods60 rats were randomly divided into 5 groups equal in number. Group A was control group. Sensorineural deafness was induced in animals of Group B, C, D and E. Bone marrow NTCSCs transfected by green fluorescent protein （EGFP） gene were transplanted to left ear cochlea modiolus of rats in group C, D and E. The cochlear axis slice was observed under a fluorescence microscope after HE staining. The survivorship, distribution site and expression of NTCSCs transfected with EGFP were observed.ResultsIn the slices of inner ear cochlea, the bone marrow NTCSCs transfected by green fluorescent protein （EGFP） gene were dispersed in the lacuma of the tympanic canal of cochlea in group C （one week after transplantation）, in the basal membrane and Corti’s organ in group D （two weeks after transplantation）. In group E （four weeks after transplantation）, aggregation of NTCSCs cluster with two or three cells was observed, and the location was close to basal membrane and Corti’s organ with good distribution and tendency of migratory behaviour. With the time extension of bone marrow NTCSCs transplantation, Nestin （＋） cells significantly increased （P〈0.05） while Myosin Ⅶa （＋） showed no significant change in number of cells （P〉0.05）.ConclusionsBone marrow NTCSCs transplanted to rat cochlea can gradually differentiate into spiral ganglion neurons, and therefore can repair acoustic nerve injury to some degree.