The Effect Of Echinacoside On Proliferation And Of Neural Stem Cells In Vitro

Objective:This topic through the establishment of fetal rat neural stem cell culture model, giving the echinacoside intervention, to observe the effect of echinacoside on neural stem cell self-renewal and multilineage differentiation potential, lay the foundation for the future research and development of neural stem cell protection drugs.Method:Take the primary neural stem cells on the 14.5 day pregnant rat. Primary neural stem cells were cultured, passaged in vitro isolation, and carried on Nestin immunofluorescence identification. Take neural stem cells of seventh days for experiment. Cells were cultered in 96-well plates at a concentration of 1x104 for 24h,then intervent with different concentrations of echinacoside for 24h and 48h.Concentrations of 0.021、0.043、0.087mmol/L were seted low, medium and high dose group intervened for 24h for subsequent experiments. Neural stem cells were randomly divided into normal group, low, medium and high dose groups. In addition to the normal group, low, medium and high dose groups replaced with a final concentration of 0.021、 0.043、0.087mmol/L for 24h. The proliferation of neural stem cells was measured by immunofluorescence labeling for BrdU.The protein expression of GDNF, GFRal, Ret was determined by Western blot.Results:The primary neural stem cells were isolated and cultured in vitro, cells suspended in culture medium, more appear in pairs,2～3 days of training, ranging from the size of the visible mass of cells, cell edge bright, strong refraction, was suspended state, namely neural ball. The cultured cells by Nestin immunofluorescence staining, under fluorescent microscopy showed strong red fluorescence, indicating that they are expression of neural stem cell specific antigen Nes. MTT results showed that in a certain range, adding different concentrations of echinacoside (0.010-0.175mmol/L) on cell toxicity, improve the survival rate of cells. BrdU immunofluorescence showed that, compared with the normal group, there was significant difference of echinacoside in each group (P<0.05). Western blot showed, compared with the normal group, the echinacoside dose group could increase GDNF, GFRal, Ret protein expression in different degree, the difference was statistically significant (P<0.05).Conclusion:1 Echinacoside has neuroprotective effect, improving the survival rate of the neural stem cells, and showed dose effect relationship;2 Cells of each group BrdU showed that echinacoside can promote the proliferation of neural stem cells in vitro, and showed dose effect relationship;3 Cells of each group WB showed echinacoside can promote glial cell neurotrophic factor (GDNF) and its receptor GFR - Ret expression increased, and showed dose effect relationship; to promote the positive effect of neural stem cells differentiation in vitro.4 In summary, the results indicated that the effect of the effect of the growth and differentiation of the neural stem cells in vitro was positive.