ABSTRACT
BACKGROUND: Bone marrow mesenchymal stem cell transplantation is superior to neural stem cell transplantation to repair spinal cord injury; however, the therapeutic effect is unstable and possibly related to microenvironment.OBJECTIVE: To study the differentiation of cultured in vitro bone marrow mesenchymal stem cells (BMSCs) into neurocytes by establishing a microenvironment and to observe differential expression of protein.DESIGN, TIME, AND SETTING: Observational contrast study was performed at the Laboratory of Neurobiology, Basic Medical College, Harbin Medical University from July 2005 to May 2007.MATERIALS: Adult Wistar rats and newborn fetal rats were used in this study.METHODS: Spinal cord was obtained from fetal rats to culture neurocytes. While, BMSCs were separated from bone marrow of adult rats, and they were then cultured in vitro, proliferated, and labeled with red fluorescin PKH26. BMSCs and neurocytes were individually cultured in the BMSCs group and the neurocyte group, respectively. In addition, BMSCs and neurocytes were co-cultured in vitro in double-layer culture dish in the co-culture group and the layered combination group, respectively.MAIN OUTCOME MEASURES: The obtained cells after 7-day culture were immunofluorescently detected by neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP). Surface enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS) technique was used to analyze associated protein that was apparently changed during the differentiation from BMSCs into neurocytes.RESULTS: Seven days after co-culture, BMSCs were morphologically shared like neurocytes. Immunofluorescence indicated that NSE- and GFAP-positive ratios of BMSCs in the co-culture group were significantly higher than the layered combination group (P < 0.05); while, the ratios in the layered combination group were significantly higher than BMSCs alone group (P < 0.05). Five protein expressions were changed during the differentiation from BMSCs into neurocytes, for example, TIP39_RAT and CALC_RAT expressions increased in the layered combination group, which were 5.344 and 2.805 times as the primary expressions; INSL6_RAT, PNOC_RAT, and PCSKI_RAT expressions decreased, which were 0.380, 0.499, and 0.437 times as the primary expressions.CONCLUSION: By a microenvironment, both BMSCs and neurocytes in the co-culture and layered combination groups can differentiate into neuroblasts; while, contact differentiation ratio is higher than non-contract one. The differentiation is closely related to five proteins, including TIP39_RAT, CALC_RAT, INSL6_RAT, PNOC_RAT, and PCSK1_RAT.