The Role Of MAPK In The Signal Transduction Mechanism Of The BMMSCs Transdifferentiation

Introduction：There are many ophthalmic diseases leading to blindness such as ischemic opticneuropathy，optic neuritis and optic atrophy, etc. There are no enough effective measures totherapy them up to now. The common pathological mechanism of these diseases is that thedisfunction and/or degeneration arise in the optic nerve and retinal neural cells. The mainaim is to protect the optic nerve from the injured factors and support the retinal neural cells toregenerate. Recently, the developing research on stem cells and gene technique provides to usnew pathway to therapy patients with the ophthalmic diseases.The bone marrow mesenchymal stem cells (BMMSCs) are a kind of adult stem cells inthe bone marrow besides haemopoietic stem cells. The BMMSCs originate from themesoderm in early period of embryogenesis, and they have the self-replication ability and thepotentiality to multi-directional differentiation. The BMMSCs can differentiate into bone, fatand cartilage cells of the mesodermal lineage. In addition, they are able to transdifferentiateinto muscle, lung and gut epithelial cells of the endodermic lineage as well as epithelial cellsand neurons of the neuroectodermic lineages. The sudy of BMMSCs transdifferentiation intoneuron-like cells is a hot spot in the adult stem cells research lately. Though we find thatBMMSCs can differentiate into neuron-like cells with the help of some inducers, the otherpossible mechanism is still unclear. The researchers attempt to figure out the more confirmedmechanism of BMMSCs neural transdifferentiation and obtain a mass of nerve seed cells tocure these disorders by cell therapy.Objectives: We explore the possible function of the proteins related with MAPK (ERK, P38andJNK) during the period of BMMSCs transdifferentiation into neuron-like cells with the helpof brain-derived neurotrophic factor (BDNF), and figure out the possible mechanism ofBMMSCs transdifferentiating into the neural cells.Materials and methods:Rat BMMSCs were isolated and cultivated by combination methods of plasticadherence method and digestion control in vitro. We filtered the cells in good state to furtherresearch after identifying cell surface markers for the cultivated BMMSCs using flowcytometry (FCM) and immunofluorescence (IF) and designing growth cruve of BMMSCswith MTT. IF was applied to examine the expression of the neuronal specific makers (nestin,NeuN and MAP-2) after BMMSCs were induced by BDNF. The proteins related with MAPK(ERK, P38and JNK) were detected during the different phases of BMMSCstransdifferentiation into neuron-like cells by Western Blotting.Results：We performed FCM analysis on the cells of passage3. BMMSCs expressed both CD90and CD44(positive ratio are97.7%and74.4%respectively). In contrast, these cells werenegative for CD11b and CD45(negative ratio are0.12%and0.11%respectively). Theexpression of CD90and CD44were further confirmed by IF. Therefore, there was noevidence of hematopoietic precursors in the cultures.The induced cells adopted the morphological features typical of neurons such asrefractile cell bodies and long branching processes with growth cone-like terminal structures.IF showed that the induced cells expressed both immature neuronal markers (nestin and NeuN)and mature neuronal markers (MAP-2). The nestin expression dropped along with theinduction. The expression of12hours after induction is higher than that of3days and1 weeks after induction (p＜0.001), and the expression of3days after induction is higher thanthat of1weeks after induction (p＜0.001). The NeuN expression of12hours after inductionis lower than that of3days and1weeks after induction (p＜0.001), and the expression of3days after induction is higher than that of1weeks after induction (p＜0.05). The MAP-2expression elevated along with the induction. The expression of1weeks after induction ishigher than that of12hours and3days after induction (p＜0.001), and the expression of3days after induction is higher than that of12hours after induction (p＜0.001).We detected the proteins related with MAPK (ERK, P38and JNK) during the differentphases of BMMSCs transdifferentiation into neuron-like cells using Western Blotting. Theexpressions of the p-c-Raf, p-MEK and p-ERK1/2after induction are exclusively higher thanthat before induction. There are no differences in the expression of p-p38and p-JNK betweenbefore and after induction.Conclusion:Rat BMMSCs can be isolated and cultivated in vitro. BMMSCs induced by BDNF notonly adopted the morphological features typical of neurons but also expressed the specificmakers for neuron, i.e. nestin, NeuN and MAP-2, so the induced cells may be neuron-likecells. During the different phases of BMMSCs transdifferentiation into neuron-like cells, theexpression of p-c-Raf, p-MEK and p-ERK after induction is apparently higher than thatbefore induction. Therefore, Raf MEK ERK1/2signal transduction could be veryimportant during the neural differentiation.