The Study Of Differentiation Of Neural Stem Cells In Vitro And Function Of Neural Stem Cells In Ischemic Brain Promoted By Ginsenoside Rg1

Objective1. To observe the effect of ginsenoside Rg1 on proliferation differentiation of neural stem cells.2. To observe the effect of ginsenoside Rg1 on promotion of the function of neural stem cells in brain after focal cerebral ischemia in rats.3. To explore the possibility about treatment of ischemic cerebral vasculardisease using the proliferation and differentiation of neural stem cells induced by ginsenoside Rg1.Methods1. First, forty neonatal Wistar rats were used for these experiments as follows: neural stem cells primary culture, the proliferation of neural stem cells induced by ginsenoside Rg1 in the concentration of 60 mg/L, the influence of ginsenoside Rg1 on proliferation in different concentrations of 40 mg/L, 80 mg/L and 120 mg/L, as well as the influence on differentiation of neural stem cells in different concentrations of 20 mg/L 40 mg/L and 80 mg/L .Non-serum culture method was used to culture neural stem cells from cortex and hippocampus in the brain .Neurospheres were counted and A value was detected by MTT. To observe the proliferation and differentiation of neural stem cells, we identified nestin(the marker of neural stem cells),BrdU(the marker of proliferating cells) ,NSE positive cells, GFAP positive cells and GalC positive cells by immunocytochemical and immunofluorescence technique.2. Then, sixty adult male Wistar rats were divided into sham group, ischemia group and ginsenoside Rg1 treatment group. Focal cerebral ischemia was produced by an intraluminal filament occlusion of the MCA. Neurologic deficit was scored as described previously by Hata et al . The thymidine analog bromodeoxyuridine(BrdU) was administered once every four hours intraperitoneally(50mg/kg,Sigma,USA) for four times before execution of the animal to label proliferating cells. By immunohistochemical single statining and double-immunofluorescence technique,the number of BrdU ,nestin,NSE and GFAP positive cells, as well as nestin/BrdU,NSE/BrdU and GFAP/BrdU double-labelled positive cells were counted. The effects of ginsenoside Rg1 on the immunoreactivity for them at 1d, 3d, 7d and 14d after focal cerebral ischemia were also observed in comparison to controls. The results were examined by student's t test and p less than 0.05 was considered significant.Results1. Neural stem cells were isolated and cultured from cortex and hippocampus of the brain in the neonatal rats with success under this experimental condition.2. Ginsenoside Rg1 in the concentration of 60 mg/L promoted the proliferation of neural stem cells in vitro. There were significantly increase of the number of neurospheres and the A value in the concentration of 40 mg/L and 80 mg/L of ginsenoside Rg1 treatment group ,whereas decreased in concentration of 120 mg/L treatment group.3. Ginsenoside Rg1 supplementation group displayed a significant decrease in nestin positive cells during differentiation whereas showed an increase of NSE, GFAP and GalC positive cells compared with control group, and the NSE positive cells increased in a dose-dependent manner at sameplating densities.4. We made focal cerebral ischemia successfully by an intraluminal filament occlusion of the right MCA.5. The neurologic deficits of model rats were improved after pretreatment with ginsenoside Rg1.6. The positive cells of nestin, BrdU,NSE and GFAP, as well as the double-labeled positive cells of nestin/BrdU,NSE/BrdLU GFAP/BrdU were significantly increased after the rats were given ginsenoside Rg1, compared with the control.Conclusions1. These results indicate that ginsenoside Rg1 can promote the proliferation and differentiation of neural stem cells in vitro.2. Pretreatment with ginsenoside Rg1 improved the neurologic deficits of focal cerebral ischemia.3. The proliferation and differentiation of neural stem cells can be induced by ginsenoside Rg1 after focal cerebral inchemia in rats. It suggest that ginsenoside Rg1 can promote the function of neural stem cells of the brain after focal cerebral ischemia, which contribute to the theoretical basis for treatment of ischemic cerebral vascular disease.