The Experimental Study Of Culture Of Bone Marrow-derived Mesenchymal Stem Cells In Vitro And Cochlear Transplantation

Objective:The idea of our experiment was to establish the methods that separate, culture, identify and label SD rat bone marrow stem cells, and to observe the survival, distribution and differentiation of labeled BMSCs after transplantation into damaged by drug SD rat cochlea. In order to provide scientific basis for BMSCs transplantation therapy of sensorineural hearing loss.Methods:1. Isolation, culture, purification, identification of BMSCs:The SD rat bilateral femur and tibia were extracted and bone marrow stem cells were cultured by adherence screening method. Then the cells were serial subcultured and expanded. The changes of cell shape and growth characteristic were observed under inverted phase contrast microscope. The expression of BMSCs phenotypic markers (CD29,CD90,CD117,CD34,CD45) were identified by immunocytochemistry.2. To establish and identification sensorineural hearing loss SD rat model:Rats were treated hypodermically with neomycin for 12 days. Auditory brainstem response(ABR) were used to identify the model.3. Labeled BMSCs::The fourth generation of BMSCs were labeled with CM-Dil.4. BMSCs cochlear transplantation:BMSCs were transplated into cochlea of drug-damaged deafened SD rats via round window. After two weeks, the animals were killed. The survival, distribution and differentiation of engrafted BMSCs were observed by immunofluorescence method.Results:1. Adherent cultured BMSCs expressed CD29,CD90,CD117 instead of CD34,CD45 by immunofluorescent staining and consistent with the characteristics of SD rat BMSCs. 2. After rats were treated hypodermically with neomycin for 12 days, audibility threshold of SD rats could reach more than 80dB.3. After BMSCs were transplated into cochlea of drug-damaged deafened SD rat via round window, the cells could survive more than two weeks, mainly locate in basic turn, less in the middle turn and top turn.4. After transplantation, the BMSCs were observed to express the specific markers of inner ear hair cells:Myosin7a and Math1.Conclusions:1. Adherent culture could isolate and subculture the pure BMSCs.2. Application of neomycin can be established an ideal deadness animal model for transplantation.3. CM-Dil can be used as a tracer marker for BMSCs in vivo.4. BMSCs can be survive more than two weeks in the SD rat cochlea of drug-induced deafness and differentiate into hair cell-like cells.