Gene expression and calcium ion concentration variation in bone marrow mesenchymal stem cells differentiating into neuron-like cells / 中国组织工程研究

ABSTRACT

BACKGROUND: Monosialotetrahexosylganglioside plays an important role in a variety of physiological processes, such as the nerve cel growth and development, differentiation, regeneration and intracel ular and extracel ular information transmission. OBJECTIVE: To investigate the effects of monosialotetrahexosylganglioside on the changes of gene expression and calcium ion concentration in the course of inducing the mesenchymal stem cells into neuron-like cells with Woodbury’s method. METHODS: The mesenchymal stem cells from Sprague Dawley rats were cultured after isolated and purified, After 5 passages in culture, the cel integrated into a dense monolayer, and treated with 50 mmol/L monosialotetrahexosylganglioside for 24 hours as the monosialotetrahexosylganglioside group; then the mesenchymal stem cells were induced into neuron-like cells with the methods of Woodbury after pre-cultured for 24 hours, and set the control group. The protein and mRNA expression levels of growth-associated protein 43, neuron-specific enolase, neurofilament and nestin were detected by immunocytochemistry and real-time PCR, respectively. The fluorescence intensity of intracel ular free calcium ion before and after inducing was detected by laser scan confocal microscope. RESULTS AND CONCLUSION: After induction, the expression levels of growth-associated protein 43, neuron-specific enolase, neurofilament and nestin of the mesenchymal stem cells in the monosialotetrahexosylganglioside group were higher than those in the control group (P < 0.05), demonstrating that monosialotetrahexosylganglioside could promote the differentiation of mesenchymal stem cells into neuron-like cells. The fluorescence intensity in mesenchymal stem cells was increased gradual y in two groups after the medium was replaced by the induction medium, attained its peak value at 100 seconds and then decreased gradual y, but the fluorescence intensity was stil higher than that before the induction at 20 minutes. The fluorescence intensity of intracel ular free Ca2+ was increased significantly in the monosialotetrahexosylganglioside group when compared with the control group (P < 0.05), suggesting that monosialotetrahexosylganglioside could increase the concentration of intracel ular free Ca2+, and intracel ular free Ca2+ may be useful in the course of induction. The changes of protein expression levels of growth-associated protein 43, neuron-specific enolase, neurofilament and nestin were not significant after induction, indicating that Woodbury classic induced programme could regulate the post-transcriptive protein level.