Study On The Effect Of Rat MSCs Differentiat Into Neuron-like Cells And The Expression Of SMNmRNA In Neuron-like Cells Inducing By Salvia Mitiorrhiza And Veratrum Nigrum

Objective:Mesenchymal stem cells(MSCs) were isolated from the Spragu-Dawley rats and cμlutred in vitro,then to study the biological characteristics of MSCs.Compairing the effects of that MSCs were dieffrentiated into neuron-like cells,which inducing by the decoction and drug-containing serum of Salvia Mitiorrhiza,Veratrum nigrum.To investigate the expression of SMNmRNA in neuron-like cells,which inducing by the decoction Salvia Mitiorrhiza and Veratrum nigrum,and to analyse the mechanism of them.To explore the ideal factors which can elevate the differentiation percentage of MSCs dieffrentiated into neuron-like cells,and also to explore the ideal condition which can enhance the expression of SMNmRNA in neuron-like cells.It might offer reference for transplanting neuron-like cells to treat spinalm uscμlarat rophy(SMA).Methods:1.To investigate the effects of MSCs were isolated from the rats by means of the density-gradient centrifugation and the differential time adherent method.The stable and uniformal MSCs were instaurating in vitro.To observe the morphologic characteristics of MSCs,MTT method was used to investigate the proliferation activity of MSCs,the growth curve of MSCs was drawing according as proliferation activity of MSCs.The cell surface antigens and Cell cycle of MSCs were detected by flow cytometry(FCM).2.The effects of that MSCs were dieffrentiated into neuron-like cells induced by control group, classic group,Salvia Mitiorrhiza,Veratrum nigrum,Salvia Mitiorrhiza and Veratrum nigrum,was investigated,To explore the most effective concentration of the above groups on differentiation MSCs into neuron-like cells.Neuron-specific enolase(NSE),Nestin,gliall fibrillary acaidic protein(GFAP) of neuron-like cells were detected by immunocyto chemistry.The morphologic characteristics and the proliferation of neuron-like cells was observed.3.FQ-PCR was used to detect the expression of SMNmRNA in neuron-like cells,which inducing by control group,classic group,Salvia Mitiorrhiza decoction,Veratrum nigrum decoction, Salvia Mitiorrhiza and Veratrum nigrum decoction,To analyse expression of SMNmRNA in the above groups.4.To prepare different concentration serum and drug-containing serum(control group serum, Salvia Mitiorrhiza serum,Veratrum nigrum serum,Salvia Mitiorrhiza and Veratrum nigrum serum).To explore the the most effective concentration of the above groups on differentiation MSCs into neuron-like cells.NSE,GFAP,GFAP of neuron-like cells were detected by immunocyto chemistry.The morphologic characteristics and the proliferation of neuron-like cells was observed.Results:1.The MSCs isolated with differential time adherent method has characteristic of high homogenize,proliferating actively and differentiating quickly.The results of the surface antigens of MSCs detected by flow cytometer showed the positive expressions of CD29 and CD44 and the negative expressions of CD45,It is accord with the immunological characteristic of MSCs, It's also substantiated that MSCs coμld be obtained and purified in vitro by our experimentation..2.The most effective differentiation concentration ofβ-mercaptoethanol(β-ME),Salvia Mitiorrhiza,Veratrum nigrum,Salvia Mitiorrhiza and Veratrum nigrum were L-DMEM cμlture medium containing 1mmol/1β-mercaptoethanol,L-DMEM cμlture medium containing 10% fetal calf seurm(FCS) and 120μl/ml Salvia Mitiorrhiza,L-DMEM cμlture medium containing 10%fetal calf seurm(FCS) and 140μl/ml Veratrum nigrum,L-DMEM cμlture medium containing 10%FCS and 80μl/ml Salvia Mitiorrhiza and Veratrum nigrum respectively.The induced expression rate of NSE and Nestin in the induced groups was higher than that in the control group,P＜0.01;The expression rate of Nestin in Salvia Mitiorrhiza and Veratrum nigrum was higher than that inβ-ME;The expression rate of NSE in Salvia Mitiorrhiza and Veratrum nigrum was higher than that inβ-ME,P＜0.05;but negatively with GFAP.3.The most effective differentiation concentration of Salvia Mitiorrhiza serum,Veratrurn nigrum serum,Salvia Mitiorrhiza and Veratrum nigrum serum were L-DMEM cμlture medium containing 10%fetal calf seurm(FCS) and 20μl/ml Salvia Mitiorrhiza serum,L-DMEM cμlture medium containing 10%fetal calf seurm(FCS) and 25μl/ml Veratrum nigrum serum,L-DMEM cμlture medium containing 10%FCS and 15μl/ml Salvia Mitiorrhiza and Veratrum nigrum serum respectively.The induced expression rate of NSE and Nestin in the induced groups was higher than that in the control group,P＜0.05;The expression rate of Nestin in Salvia Mitiorrhiza and Veratrum nigrum serum was higher than that inβ-ME;The expression rate of NSE in Salvia Mitiorrhiza and Veratrum nigrum serum was higher than that inβ-ME,P＜0.01;but negatively with GFAP.4.The relative expression abundance of SMNmRNA inβ-ME,Salvia Mitiorrhiza,Veratrum nigrum,Salvia Mitiorrhiza and Veratrum nigrum was higher than that in control group,P＜0.01.The relative expression abundance of SMNmRNA in Salvia Mitiorrhiza and Veratrum nigrum was higher than that inβ-ME,P＜0.05.Conclusion:1.The differential time adherent method was a ideal method to isolate MSCs from Spragu-Dawley rat.2.Salvia Mitiorrhiza decoction,Salvia Mitiorrhiza and Veratrum nigrum decoction coμld induce MSCs to differentiate into neuron-like cells in vitro.The effects of that MSCs were dieffrentiated into neuron-like cells induced byeratrum nigrum decoction was indistinctively. The differentiation effects of Salvia Mitiorrhiza and Veratrum nigrum decoction was higher than that in other groups,The reasons might be that there were some combination components and effectiveness in the Salvia Mitiorrhiza and Veratrum nigrum decoction,the contrary integration effects of Salvia Mitiorrhiza and Veratrum nigrum decoction need to be explore profoundly.3.The relative expression abundance of SMNmRNA in Salvia Mitiorrhiza and Veratrurn nigrum decoction was higher than that in other groups.The reasons might be that the SMN gene was in state of silencing,there was no transcription and expression of SMNmRNA.It's might be the components and effectiveness in Salvia Mitiorrhiza and Veratrum nigrum decoction that activated the SMN gene of neuron-like cells,and enhanced the expression of SMNmRNA.4.The differentiation effects of Salvia Mitiorrhiza and Veratrum nigrum serum was lower than that in Salvia Mitiorrhiza and Veratrum nigrum decoction.The reasons might be that the induced components and effectiveness in Salvia Mitiorrhiza and Veratrum nigrum decoction were weaken by through the metabolism of rats.