Experimental Study On Induced Differentiaion Of Rat Mesenchymal Stem Cells Into Neural Cells And The Expression Of MASH-1 Invitro

ObjectiveTo investigate the changes of calcium ion concentration and gene expression in mesenchymal stem cells(MSCs)during their differentiating into neuron-like cells induced byβ-ME and MASH-1in vitro which can make a further research for the truth of inducing the mesenchymal stem cells into neurons.Methods1. MSCs were obtained by gradient centrifugation on percoll(1.073g/ml) from femurs and tibias of adult SD rats aseptically. Then through washing with 5ml DMEM and discarding non-adherent cells, MSCs were purified and expanded. Invert phase-contrast microscopy was used to observe morphology change of MSCs each day.2. For neural differentiation,MSCs(P5,P10,P15)were induced withβ-ME.The treatment protocol: MSCs were pretreated with preinduction media consisting of L-DMEM + 1mmol/Lβ-ME + 20%FBS for 24h. Then preinduction media were removed and two groups of neuronal induction media were added. First group: L-DMEM + 5mmol/Lβ-ME; second group: L-DMEM + PBS. Invert phase-contrast microscopy was used to observe morphology change of MSCs after induction.3. The differentiated MSCs were identified by means of immunocytoche -mistry for neural-specific markers: NESTIN,NSE,NF,GFAP after induced for 1h and 5h.4. Nissl Body staining was used to identified the differentiated MSCs after induced.5. We constructed the intervention carrier which can express MASH - 1 for the small interfering RNA(siRNA), then transfected into MSCs. And transfected interference group(with the no righteous sequence siRNA)and negative control group(with the PBS).6. Test the changing of the expression of MASH - 1 induced or not.Results1. MSCs adhere to the flask,most of which appear fibroblast-like.Their character was stable and the morphologies of MSCs had no change after increasing passages. The down generation cells can proliferation faster than original generation cells. The cells can reach confluence after 7-9 days.2. After induced by L-DMEM + 5mmol/Lβ-ME, cells body of most MSCs contract and exhibit spherical, with increasing thin processes.3. Non-induced MSCs show negative stains for NESTIN,NSE,NF,and GFAP. After inducement, NESTIN,NSE,NF and GFAP expressions raise apparently. Induced passage 5 MSCs showing strong or moderate NESTIN,NSE,NF,GFAP are 82.25±1.59%, 85.79±2.79%, 80.71±5.68%, 45.71±3.12% respectively, which is consistent to the 10th and 15th passage and statistical analysis showed that the differences among three different passages were insignificant(P>0.05).4. Induced cells with typical neron-like morphology have large quantity of Nissile body in the cytoplasm.5. Real-time PCR found that MASH-1-mRNA expression of MSCs cells in the transfected group was 0.0947±0.00854 on average which was much lower than that of the blank group 0.4693±0.01823 and negative control group was 0.4583±0.02141 . The difference was of obviously statistical significance (P < 0. 01).Conclusion1. Percoll density gradient centrifugation combined with adherence is a well-referenced way for the separating and purifying of MSCs.2. MSCs can be differentiated into neuron and neuroglia cells in vitro byβ-ME. 3. The differentiated MSCs,which Nissl Body staining was positive,had some of the characteristics of mature neuron in structure.4. It shows the MASH-1 for siRNA can suppress the MASH-1 gene expression effectively that the Real-time PCR tests have revealed a Real– the transfected group cells'MASH-1 express quantity significantly reduced.