The Effect Of Let-7d On The Differentiation Of Bone Marrow Mesenchymal Stem Cells Into Neurons

Background and objective Micro RNAs is a kind of highly conservative, endogenous non-coding small RNA molecules, which play an important role in the growth and development of the normal body and disease. Let-7 family members play an important role in differentiation of the bone marrow mesenchymal stem cells(MSCs). To understand the influence of let-7d to the MSCs differentiation to neural cell can promote the development of stem cell transplantation in intracranial. In this paper, we explore the role of the let-7d in the differentiation of MSCs to neuronal cell by constructing let-7d lentiviral vector.Materials and methods 1 Build the rat lentiviral vector, that is transfection blank lentiviral vector, transfection let-7d purpose gene lentiviral vector, the let-7d inhibition gene lentiviral vector, and then transfection the rat MSCs using these lentiviral vector. According to transfection lentiviral vector, the experiment is divided into four groups: namely not transfection groups, negative control groups(transfection blank lentibiral vector), up transfection groups(transfection let-7d-LV), down transfection groups(transfection let-7d-inhibition-LV). 2 To detect the survival rate of the transfection cells by MTT method.3 To induce the transfection cells to neurons, by put in the fasudil in the transfection cells.4 Using the immunocytochemistry method to tests the expression of neuronal markers NSE and MAP-2 in the differentiation cells, to make sure MSCs have been differentiated into neurons;5 To use the RT- PCR method detects MAP-2 m RNA expression of the differentiation cells, to explicit the differentiate efficiency of MSCs to neurons.Results 1 Under the inverted fluorescence microscope we found the differentiate MSCs release fluorescent display, prompt the let-7d lentiviral vector transfection was successed. There is not obvious difference of the cell fluorescence intensity between groups. That is there is not obvious difference of the cell transfection efficiency between different groups.2 The cell survival rate was detected by MTT method and found that the cell surviral rate has no significant difference among the negtive control groups, up transfection groups, and down transfection groups(P >0.05). While the cell survival rate of the three groups were all lower than not transfection groups.3 Under the microscope, we found that cell morphological changes, after join the fasudil in the transfection cells of different groups. Immunocytochemistry method found high expression of neuronal markers NSE and MAP-2, indicated that fasudil can induce the differentiation of MSCs to neurons.4 The immunocytochemistry method found that the NSE and MAP-2 fluorescence intensity was higher in the up transfection group than that in the down transfection groups and negtive control groups(P < 0.05). And the NSE and MAP-2 fluorescence intensity was lower in the down transfection group than that in the up transfection groups and negtive control groups(P < 0.05).5 RT-PCR and Western blot method detected the MAP-2 m RNA expression, found that the MAP-2 m RNA expression quantity was higher in the up transfection groups than in the down transfection groups and negtive control groups(P < 0.05), and the MAP-2 m RNA expression quantity was lower in the down transfectiongroups than in the up transfection groups and negtive control groups(P < 0.05).Conclusion 1 Let-7d lentiviral vector can transfect rat MSCs in vitro, and the transfected MSCs can be used for experimental study.2 Let-7d can promote the MSCs differentiated into neurons, and by controlling the let-7d expression can influence the differentiation efficiency of MSCs into neurons.