| ObjectiveTo establish a model of in vitro aging of neural stem cells induced by D-galactose.After ginsenosides interfered with aging neural stem cells,quantify the expression changes of two endogenous transcription factors SOX2 and BMI-1 in neural stem cells,and explore the maintenance and enhancement of ginsenosides,the role of aging neural stem cell biological characteristics.Methods1 Rats aged 15-17 days were selected and primary NSCs were obtained by mechanical isolation of embryonic hippocampal NSCs,followed by subculture.NSCs were identified by the specific marker Nestin using immunofluorescence staining.2 CCK-8 method to screen the optimal concentration and optimal time of D-galactose-induced neural stem cell in vitro aging model.Set 6 different time points(6h,12h,24h,48h,60h,72h),plant 6 plates,set Dmem group,control group and different dose groups in each plate(5,10,15,20,25,30,35,40mg/ml),then add CCK-8,after 1-4h,measure the absorbance at 450nm with a microplate reader,organize the data,and draw a standard curve.3 Using the MTS method to detect the proliferative capacity of NSCs by MTS method,the optimal dosage of ginsenoside(lug/ml)and the best time(24h)were tested.The ginsenoside-induced D-galactose-induced nerve was detected by CCk-8 method.The effect of aging neural stem cells in a stem cell in vitro aging model.4 Western Blot method was used to detect the expression of SOX2 and BMI-1 protein in neural stem cells after ginsenoside treatment of aged neural stem cells.The experiment was divided into normal group,aging group and aging plus ginsenoside group.Proteins were first extracted from the three groups of NSCs,and the protein samples were prepared according to the instructions of the protein extraction kit.SDS-PAGE electrophoresis,application of the primary antibody,and finally the gel imager was used to detect the target protein.Image Pro Plus 7.0 was used.The software analyzes and counts the strip results.5 Immunofluorescence and nuclear staining were used to detect normal neural stem cells,aging neural stem cells and ginsenosides.After aging neural stem cells,the specific markers in neural stem cells,Nestin,and the expression of SOX2 and BMI-1 genes.The experiment was divided into normal group,aging group,aging plus ginseng saponin group.After cell culture,modeling,adding ginsenoside and performing preliminary immunofluorescence routine experiment,the corresponding Nestin,BMI-1 and SOX2 primary antibodies were added.After DAPI staining,after sealing,fluorescently labeled cells were observed under a laser confocal microscope and photographed.Image proplus image analysis software was used to analyze the number of cells,areal density and optical density of positive cells in the photographs.Results1 The embryonic rat hippocampal NSCs were successfully isolated and cultured,and the NSCs-specific marker Nestin was expressed by immunofluorescence.The primary culture and subculture were performed,and the purity was 95%or more of NSCs.2 The optimal concentration of D-galactose-induced neural stem cells in vitro aging model is 35mg/ml,and the optimal time for D-galactose-induced neural stem cell in vitro aging model is 24h.3 Using CCk-8 method to detect the effect of ginsenosides on aging neural stem cells in D-galactose-induced neural stem cells in vitro aging modelCompared with the normal group,the absorbance of the aging group decreased significantly,and the difference was statistically significant(P<0.01).Compared with the aging group,the absorbance of the ginsenoside group increased significantly,and the difference was statistically significant(P<0.01).4 Western Blot assay for the expression of SOX2 and BMI-1 protein in neural stem cells after ginsenoside treatment of aged neural stem cellsThe SOX2 and BMI-1 proteins in the normal group of neural stem cells,the aging group and the aging plus ginsenoside group were positively expressed.Compared with the normal group,the expression of SOX2 in the neural stem cell aging group was significantly lower(p<0.01).The expression of SOX2 was higher in the neural stem cell aging plus ginsenoside group than in the aging group.The difference was statistically significant.(p<0.05).Compared with the normal group,the expression of BMI-1 in the neural stem cell aging group was significantly lower(p<0.01).The expression of BMI-1 was significantly higher in the neural stem cell aging plus ginseng saponin group than in the aging group.Statistically significant(p<0.01).5 Immunofluorescence and nuclear staining to detect normal neural stem cells,aging neural stem cells and ginsenosides after aging neural stem cells,neural stem cells specific markers Nestin,and SOX2 gene and BMI-1 gene expressionNestin,SOX2,and BMI-1 were positively expressed in the normal NSCs group,the NSCs aging group,and the NSCs aging plus ginsenoside groups.Compared with the normal group of NSCs,the Nestin,SOX2,BMI-1 areal density,optical density and positive cells of NSCs aging group were significantly decreased,the difference was statistically significant(P<0.01);compared with NSCs aging group,NSCs Nestin,SOX2,BMI-1 areal density,optical density and number of positive cells in the ginsenoside group increased,the difference was statistically significant(P<0.05 or P<0.01)ConclusionThe optimal concentration dose of D-galactose-induced neural stem cell in vitro aging model is 35 mg/ml,and the optimal time is 24h.Ginsenoside can promote the expression of SOX2 and BMI-1 in aging NSCs,which proves that ginsenoside has a certain effect on maintaining the biological characteristics of senile neural stem cells,and can participate in cell proliferation,differentiation and nerve repair of brain aging. |
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