Experimential Study For Differentiation Of Bone Marrow Stems Cells Induced By GDNF

Aims Comparing the differences of differentiation of bone marrow mesenchymal stem cells (BMSCs) induced into neuron-like cells by using glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF), further to set GDNF-GFP recombinant adenovirus virus transfected BMSCs, observing the expression of GDNF and GFP gene in BMSCs and growth and differentiation of the BMSCs after transfection.Methods Isolating and characterizing putative bone marrow derived MSCs from the Spra-gue-Dawley (SD) rats. Furthermore, differentiation effects were compared between GDNF-inducing group and BDNF-inducing group. Of these, BMSCs were isolated from the SD rats in a traditional manner, and identified based on plastic adherence, morphology, and surface phenotype assays. Besides, in GDNF-inducing group (including lOng/ml GDNF) and BDNF-inducing group (including lOng/ml GDNF), makers of neural-like cells, such as NSE and MAP-2, were detected by immunofluorescence and Western blotting at different time-point. In addition, changes of significant neural-like cells morphology and cell viability of BMSCs were observed and analyzed by statistical method during the induction with GDNF and BDNF. In ad-dition, BMSCs were transfected with recombinant adenovirus containing the GDNF and green fluorescent protein (GFP) genes. The expression of GFP was observed by fluorescence micro-scope and the expression of GDNF was detected by PCR after transfection. MTT assays were employed to determine the cytotoxicity of the transfection against BMSCs in vitro. The expres-sion of NSE and MAP-2from BMSCs were detected by immunofluorescence and Western blot-ting at different time-point after transfection.Results The result of this researches indicate that whole marrow direct adherence was a simple and convenient method for isolation and cultivation of BMSCs. BMSCs could express maker of neurons and change into significant morphology of neural-like cells by using GDNF, which could achieve more persistent and stable inducing effect than BDNF in vitro. The result of fluo-rescence microscope indicated that the expression of GFP gradually increased until3days after transfection and the GFP could be observed until14days after transfection. The result of MTT indicated that the viability of BMSCs increased compared with control group after transfection. The expression of GDNF was detected by PCR and the results demonstrated that the expression of rGDNF increased followed by teansfection until10days after transfection. Expression of the neuronal cell markers NSE and MAP-2by BMSCs was detected by immunofluorescence respec-tively5days and10days after transfection.Conclusion Whole marrow direct adherence was a simple and convenient method for isolation and culture of BMSCs. BMSCs could express maker of neurons and change into significant morphology of neural-like cells by using GDNF, which could achieve more persistent and stable inducing effect than BDNF in vitro. Furthermore, adenovirus-mediated gene transfer achieved persistent and enhanced expression of GDNF, which induced BMSC differentiation into neur-al-like cells without exogenous inducer in vitro.