The Study On The Changes Of Calcium Ion Concentration And Gene Expression In Mesenchymal Stem Cells During Their Differentiating Into Neuron-like Cells

ObjectiveTo investigate the changes of calcium ion concentration and gene expression in mesenchymal stem cells(MSCs) during their differentiating into neuron-like cells induced by genistein and U-73122 in vitro which can make a further research for the truth of inducing the mesenchymal stem cells into neurons.Methods1. Rat MSCs were separated from femur and tibia marrow by flushing out with low glucose DMEM after nipped the two ends at sterile condition, the mesenchymal stem cells were harvested after the mixed fluid was centrifugated with percoll (1.073g/ml) and then cultured. After 4 passages in culture, MSCs were divided into four groups at random, A: normal control group,B: append genistein(100umol/L) group, C: append U-73122(1umol/L) group, D: append genistein(100umol/L) and U-73122(1umol/L) group. All the four groups'cells are cultered in vitro. Observe the form and growth on the phase contrast microscope everyday.2. The mesenchymal stem cells were induced into neuron-like cells with amended methods of Woodbury's after 5 passages. MSCs were incubated in preinduction media consisting of DMEM+1mmol/Lβ-mercaptoethanol+20%FBS for 24h, then preinduction media were removed and neuronal induction media (DMEM+5mmol/Lβ-mercaptoethanol) were added for 5 hours.3. The differentiated MSCs were identified by means of immunocyto- chemistry for neural-specific markers: NSE and GFAP. The cells with brown grains were judged positive, without any colours were negative.4. After 4 passages in culture, MSCs were divided into four groups at random, A :normal control group, B: append genistein(100umol/L) group, C: append U-73122(1umol/L) group, D: append genistein(100umol/L) and U-73122(1umol/L) group. MSCs were pre-induced by 1mmol/Lβ-mercaptoethanol for 24h and then ,with the addition of L-DMEM containing 10umol/L Fluo-3/AM , for another 45min. Last , the medium was replaced with an induction medium containing 5mmol/Lβ-mercaptoethanol ,Meanwhile , we observed the changes of the fluorescence intensity and the shape of MSCs with laser beams of 488 nm wavelength.5. The total mRNA of the four groups cells were isolated before and after inducing , to mensurate the purity with ultraviolet radiation spectrophotometer. The GAP-43,NSE,NF and Nestin mRNA of four groups were expanded by Realtime reverse transcription polymerase chain reaction (RT-PCR). To analysis the outcome with SPSS ver 13.0.Results1. Most cells of four groups adhered to the flask and appeared long shuttlet-like. Their character was stable and the morphologies of MSCs had no change after passage but increased more quickly.2. After induced byβ-ME+FBS, cells body of most MSCs contract and exhibit spherical or pyramidal, with increasing thin processes, some induced cells had the neuron-like morphology. More and more cells changed after 5 hours, the cells emitted two or many poles and interweaced together, just like the typical neurons.3. The results of the immunocytochemistry indicated: the NSE and GFAP showed positive stains of four group cells, which meaned the four group cells differentiated into neuron.4. The fluorescence intensity of A group in MSCs increased gradually after the medium was replaced with the induction medium containing 5mmol/Lβ-mercaptoethanol. At 100s it attained its peak value and then decreased gradually, but at 30 minutes still higher than that before the induction. Meanwhile, we observed that MSCs differentiated into neuron-like cells. Genistein and U-73122 can decrease the increasing of intracellular free Ca2+.5. The GAP-43,NSE,NF and Nestin mRNA could be detected in four groups. They have no changes among four groups before and after inducing.Conclusion1. After MSCs was induced byβ- mercaptoethanol, the expression rates of NSE and GFAP increased obviously, cells body of most MSCs had the neuron-like morphology. It meaned MSCs could be induced into neuron-like cells byβ-ME induction.2. After MSCs was induced byβ- mercaptoethanol, The fluorescence intensity in MSCs increased gradually. It decreased the increasing of the fluorescence intensity by blocking NGF/TrkA/PLC-γsignal pathway, the intensity of intracellular free Ca2+ had no changes before and after induction. It showed Genistein and U-73122 can decrease the increasing of intracellular free Ca2+. But the expression rates of NSE and GFAP of every groups increased obviously, it showed free Ca2+ may not be the major factor.3. It can not inhibit the differentiation of mesenchymal stem cells into neurons by NGF/TrkA/PLC-γsignal pathway blockage simple. the expression rates of NSE and GFAP of every groups increased obviously, It showed the NGF/TrkA signal pathway may be not concerned with the differentiation of mesenchymal stem cells into neurons.4. The expression rates of NSE and GFAP of every groups increased, but the GAP-43,NSE,NF and Nestin mRNA have no changes among four groups before and after inducing. It showed it may be post-transcription regulation i.e. the regulation on protein level during the differentiation of mesenchymal stem cells into neurons byβ- mercaptoethanol .