| ObjectiveAmyotrophic lateral sclerosis(ALS)is a rapidly progressive neurodegenerative disease.The pathological mechanism of ALS is complex,but mitochondrial dysfunction is one of the earliest pathophysiological events and the core of pathogenesis.It has been found that conditioned medium(CM)of mesenchymal stem cells(MSCs)contained a variety of neurotrophic factors and MSC-CM had the protective effects of antioxidative and anti-apoptosis on ALS model in vivo and in vitro,which might improve mitochondrial function by regulating mitochondrial membrane potential to protect motor neurons.Ginsenosides Rgl(G-Rgl),extracted from GinSeng,a traditional Chinese Medicine,can not only improve mitochondrial function and reduce neuronal apoptosis in a variety of neurodegenerative diseases,but also promote the transduction of neurotrophic factors.Methods1.Construct ALS model in vitro by lentivirus transfectionAfter selecting the appropriate MO I value by using the lentivirus containing empty plasmid,the cells were divided into four groups:NSC34 group,vector group,SOD1WT group and SOD1G93A group,according to the different treatment with lentivirus containing empty plasmid,SOD1WT gene and SOD1G93A gene.The morphological changes of the four groups were observed,and the expression of SOD1 protein was detected by Western blot(WB)to identify the cell model.2.Treat SOD1G93A-NSC34 cells with CMThe hUCMSCs was induced into neuron like differentiation and the morphological changes were observed.The gene levels of NSE,nestin and NFM were detected by qPCR.The expression of neuron related proteins NSE,nestin and NF-M was evaluated by immunofluorescence.After successful induction,CM was extracted,in which,the contents of NGF,GDNF and BDNF were determined by ELISA.CCK8 method was used to evaluate the effects of different concentrations of CM on the proliferation and toxicity of SOD1G93ANSC34 cells.After selecting the appropriate concentration,SOD1G93A-NSC34 cells were treated by CM.According to the different treatment of cells,they were divided into three groups:Control group,SOD1G93A group and SOD1G93A+CM group.Flow cytometry was used to evaluate the effect of CM on the apoptosis rate.WB was used to evaluate the effect of CM on the apoptosis related proteins Bax,Bcl-2,Cyt c and caspase-9 of SOD1G93A-NSC34 cells.3.Treat SOD1G93A-NSC34 cells with CM combined with G-RglCCK8 method was used to evaluate the effects of different concentrations of G-Rgl and CM combined with different concentrations of G-Rgl on the proliferation and toxicity of SOD1G93A-NSC34 cells,and the appropriate combination concentration was selected for intervention.According to the different treatments,the cells were divided into five groups:control group,SOD1G93A group,SOD1G93A+CM group,SOD1G93A+G-Rgl group,SOD1G93A+CM+G-Rg1 group.Flow cytometry and WB were used to evaluate the effects of CM alone or GRgl alone or CM combined with G-Rg1 on the apoptosis rate and apoptosis protein Bax,Bcl-2,Cyt C and caspase-9 of SOD1G93A-NSC34 cells.Results1.After transfection of NSC34 cells by lentivirus with MOI value of 20,there was no significant difference in morphology between vector group and SOD1WT group overexpression cells and normal cells,whose cell bodies were polygonal,with many and obvious synapses.However,the cell shape of SOD1G93A overexpression cells became round,synapses retracted,and the cells are in poor condition,with a low confluence rate.At the same time,there was no difference in the expression of SOD1 protein between vector group and normal cells(P>0.05),but the expression of SOD1 protein in SOD1WT group and SOD1G93A group showed an over expression trend(**P<0.01).The results showed that NSC34 cell line stably expressing SOD1G93A protein was successfully constructed,which had the same protein expression and similar morphological characteristics of neurons in FALS containing SOD1G93A gene.2.After 7 days of induction,the cell bodies of hUCMSCs were convex,polygonal,synaptic elongated and neuron like.Compared with the uninduced hUCMSCs,the relative expression levels of neuron related genes NSE,nestin and NF-M in induced cells were significantly different.At the same time,immunofluorescence showed that neuron like differentiated hUCMSCs could express neuron specific proteins NF-M,NSE and nestin.It indicated that hUCMSCs were successfully induced into neuron like differentiion.In addition,ELISA assay showed that the secretion of neurotrophic factors increased significantly after neuron like differentiation(**P<0.01).According to the results of CCK8,after 50%CM was used to intervene SOD1G93ANSC34 cells,it was found that 50%CM could reduce the apoptosis rate of SOD1G93A-NSC34 cells to a certain extent(**P<0.01),and up regulate the expression level of Bcl-2 protein(#P<0.05),as well as down regulate the expression levels of Bax,Cyt C and caspase-9 protein(#P<0.05).3.According to the results of CCK8,the combination of 50%CM and 60 ug/mL G-Rgl was used to intervene SOD1G93A-NSC34 cells.It was found that compared with 50%CM or 60 ug/mL G-Rgl alone,the combination of 50%CM and 60 ug/mL G-Rgl had the most obvious anti-apoptotic effect(P<0.01),and has a stronger effect on up regulating the anti-apoptotic protein Bcl-2,as well as down regulating the pro-apoptotic protein Bax,Cyt c and Caspase9 expression level(P<0.01).ConclusionsThe neuron like differentiation of hUCMSCs can significantly increase the secretion of neurotrophic factors.CM combined with G-Rgl has obvious anti-apoptosis effect on SOD1G93A-NSC34 cells compared with CM or G-RG1 alone,and may play an anti-apoptosis effect by regulating the expression of apoptotic related proteins. |
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