Effects Of Theanine On The Proliferation And Differentiation Of Neural Stem Cells

Theanine is a kind of natural amino acid in green tea, also known as L-Theanine and N-ethyl-L-glutamine. As a kind of Non-protein amino acid, Theanine is given priority to "L" in nature and only exist in the free form. Theanine not only can be extracted from tea but also can be gained through a variety of synthetic methods. Theanine is a kind of water soluble amino acids that can easily soluble in water but insoluble in ethanol or ether. After intestinal absorption, Theanine was transported into the bloodsterm by sodium transporter, then Theanine was transported into the brain across the blood-brain barrier by leucine transporter. And some research have shown that the toxic effect of Theanine is very small. Then Clinical studies have found that Theanine could participate in the adjustment of multiple systems in the human body and possesses protective effects on the nervous system. The structure of Theanine is similar to glutamic acid, so it can be combined with glutamic acid receptors compete with glutamic acid and regulate the development of the nervous system.Neural stem cells (NSCs) is a kind of stem cells and a kind of undifferentiated cells in the brain and spinal cord. Like other stem cells, NSCs not only have have the function of proliferation and differentiation but also have ability of self-renewal, it is able to differentiate into neurons, glial cells and other types of nerve cells. Various function of neural stem cells is completed by neurons and glial cells, there are many of the central nervous system diseases, such as brain injury, cerebral hemorrhage and Parkinson’s disease. They have a lack of nerve cells or deformation of death at different degree, and thus cause nerve function damage. NSCs can repair the brain by differentiate into new neurons to replace the damaged neurons.But whether Theanine can influence neural stem cells in brain injury. So NSCs were isolated from mouse embryonic, then Theanine were add into nutrient fluid. MTS, immunofluorescent staining technique, Q-PCR and Westen blotting were used to detect the proliferation and differentiation of NSCs.1. Theisolation, culture and identification of NSCsThe isolation and culture of NSCs depends on many factors, such as animal species, age, based site, inoculation density and medium nutrition conditions. The study found that NSCs from embryonic mice are the most primitive neural stem cells compared the freshmen and adult rat, and they have capable of extended self-renewal, proliferation and differentiation. NSCs were isolated from mouse embryonic at E12.5 by mechanical blowing combined with stencils of 200 mesh. Then immunofluorescent staining technique of nestin and Brdu was used to identify the isolated neural stem cells and its capable of proliferation, and immunofluorescent staining of microtubule associated protein 2 (MAP2) and glial fibrillary acidic protein (GFAP) was used to identify the proliferation of neural stem cells. The present results suggest that neural progenitor cells from brain cortex of embryonic mice are easily extracted and naturally differentiate into neurocyte and neuroglial cells.2. Effects of Theanine on the proliferation and differentiation of neural stem cellsThe effects of various concentrations of Theanine on NSCs viability were first assessed. NSCs were incubated in the grouth medium in the presence of increasing concentra-tions of Theanine(0、1μmol/L、10μmol/L、100μmol/L、1mmol/L、 10mmol/L).MTS results showed that 100μmol/L yielded the optimal effect on cell viability. Then NSCs were divided into two groups:control group, Theanine(100μmol/L) treated group. The proliferation of NSCs were determined by BrdU labeling afer 48 hours, and differentiation of NSCs were determined by immunofluorescent staining of microtubule associated protein 2 (MAP2) and glial fibrillary acidic protein (GFAP) after 5 days. The RT-PCR and Westen blot were used to detect the expression of MAP2 and GFAP.Conclusion The optical density of Theanine (100μmol/L) treated group was significantly increased in MTS(P<0.05). The ratio of BrdU-positive cells and MAP2-positive cells against total NSCs treated with Theanine was significantly increased in comparison with that in control group(P<0.05), there was no noticeable difference in GFAP immunofluorescence between two groups. The expression of MAP2 treated with Theanine was significantly increased in comparison with that in control group, there was no noticeable difference in GFAP expression between two groups. Theanine(100μmol/L) can promote the proliferation of NSCs and its neuronal differentiation.