The Study Of Cultured Bone Marrow Mesenchymal Stem Cells Of Guinea Pig And Its Transfection With Neurotrophic Factor-3

Objective:Establishing the culture system of guinea bone marrow-derived mesenchymal stem cells (BMSCs) in vitro, and transfecting neurotrophic factor-3(NT-3) by Lipofectamin2000into BMSCs, so as to provide the foundation for the next experiments of inner ear transplantation.Methods:(1) Guinea BMSCs were extracted,cultured and subcultured by whole bone marrow directly adhering in flasks, the third generation were used for flow cytometric detection about the surface markers CD29, CD90of BMSCs and the surface antigen CD45of hematopoietic cell.(2) BMSCs of P3were transfected with pEGFP-N1-NT3by plasmid-liposome mixture which were mixed at different ratios, then calculated the transfection efficiency in48h after transfection, chose the best ratio of plasmid-liposome.(3) BMSCs of P3were transfected with pEGFP-Nl-NT3by liposome at the best ration of plasmid and liposome, then observing its fluorescent expression in1d,3D,7d,14d after transfection.(4) Extracting the protein of BMSCs in48h after transfection, validating the expression of NT-3by Western Blot.Results:(1)We got many BMSCs proliferating quickly by the method of whole bone marrow directly adhering in flasks.(2)BMSCs grew in an s-shaped curve type, largely in line with Logistic growth curve.(3)The flow cytometric detection showed the positive expression of CD29and CD90, negative expression of CD45, matching the characteristics of BMSCs.(4)The transfection efficiency was the highest with the ratio of plasmid-liposome (μg:μl) at4:12, making it the best ratio of plasmid-liposome.(5)Fluorescence was expressed strongly in48hours after transfection,and was visible but significantly decreased in7d, and could not been seen to14d.(6)NT-3gene were validated successfully expressed in BMSCs by Western Blot Conclusion:(1) BMSCs of Guinea Pig cultured by the method of whole bone marrow directly adhering in flasks high purity cells can be highly purified and proliferate quickly.(2) Eukaryotic plasmid of NT-3can be successfully mediated by Lipofectamin2000and express in BMSCs and BMSCs can be a desirable vector for gene therapy in the inner ear.