| Neural stem cell is one type of stem cells with capacity of self-renewal and can be differentiated into neurons, astrocytes and oligodendrocytes. It locates not only in the mammalian embryo nerve tissue, but also in the fetal and adult neural tissue. The finding of adult neural stem cells broad the range of neural stem cell research and provides a good germplasm resource for the cell replacement treatment of nervous central system diseases. Amyloid-βprotein is the core component of senile plaques in Alzheimer's disease, playing a key role in the pathogenic progress of Alzheimer's disease and was a crucial factor in vitro research on the treatment of Alzheimer's disease.Tea Polyphenols, called Camellia sinensis, is a flavanol substance extracted from the leaves of green tea. Since it has good antioxidant capacity and a variety of pharmacological properties. TP has been studied widely in brain ischemia, tumor, cardiovascular disease, neurodegenerative diseases, and so on. The aim of this paper is to study the neuroprotective effects of tea polyphenols from cellular level by interventing with different concentrations of tea polyphenols on Aβ25-35-induced neural stem cell injury model. And then, we study the possible protect mechanism of tea polyphenols, which will provide some basis experimental reference for using tea polyphenols to prenvent the neurodegenerative disease of central nervous system in clinical.We in vitro isolated neural stem cells from the hippocampus of E14-15d embryonic mice brain tissue and then were cultured in serum-free medium. Immunocytochemistry were performed to identify neural stem cell by using nerve cell specific surface antigen Nesti,glial fibrillary acidic protein and neuron specific enolase. Futhermore, we put different concentrations of Aβ25-35 (10,20,40 uM) into the cultured neural stem cells to induce cell damage and analyze the damage from morphology, the rate of cell survival and catalase (CAT) activity, and so on.By then, the optimal concentration was choosed. The other part of this study focus on the effect of tea polyphenols on Aβ25-35-induced injury of neural stem cells. The effect of tea polyphenols towards damaged cell were evaluate from morphology, cell viability, oxidative stress (SOD activity and MDA content) and cell apoptosis. At last, by using RT-PCR to detect the expressed apoptosis gene, we discussed the possible molecular mechanism of tea polyphenol on damaged cells.The results are as follows:1. Culture and identification of neural stem cells:Neural stem cells were isolated from the embryonic mice brain by mechanical blowing and the isolated single cells were cultured in serum free medium. There was small amount of cells gathering together in third day and a neurosperes in the seventh day. which had clear cell boundary and could be for passage. The passaged neurosperes and serum-induced neural cell were identified by immunocytochemistry staining. The results showed that the neurosperes were nestin-positive and the induced differentiation cells were respectively GFAP-positive and NSE-positive cells. Accordingly, we isolated and cultured neural stem cells with capacity of proliferation and differentiation potential.2. Aβ-induced neural stem cells damage:different concentrations of condensed Aβ25-35 (5,10,20umol/L) were co-cultured with neural stem cells. The observed morphological changes were that:smaller cell bodies,uncompleted nucleus,decreased refraction,apoptotic bodies and other apoptosis features. Giemsa staining was performed to further explain the presence of apoptotic cells. After collecting the cultured cells at 6,12.24.36,48 hours, the results of trypan blue staining to count cell viability showed that:compared with the control group, the cell viability of 10umol/LAβ25-35 group significantly decreased at 24 hour co-culture (P<0.05); at the same time, the cell viability of 20umol/L Aβ25-35 group extremely significant decreased (P<0.01). We futher detected the CAT activity of different group cells after 24 hours culture. The results showed that:compared with control group, different concertrations of Aβhad decreased the cell CAT activity, which were a certain degree of concentration dependence; cells CAT activity of 5umol/L and lOumol/L Aβ25-35 group decreased, but were not statistically significant (P>0.05); cell CAT activities of 20umol/LAβ25-35 group was extremely significant decreased (P<0.01).3. Effects of tea polyphenols on damaged neural stem cells:compared with the Aβ25-35 group, different concentrations of TP (10,20.40ug/ml) could enhance the rate of cell survival rate and protect the integrity of cell morphology, among which the protective effect of 20ug/ml TP on damaged cells were extremely significant (P<0.01). Spectrophotometry method was used to determinate the cells SOD activity and MDA content.The results show:cell SOD activity was significantly decreased(P<0.05) and MDA content increased significantly (P<0.01)in Aβmodel group; compared with the Aβ25-35 group. SOD activity increased and MDA content decreased in lOug/mlTP group. but the difference was not significant (P>0.05); cells SOD activity was extremely significant increased (P<0.01) and MDA content was decreased but not statistically significant in 20ug/ml TP group; the difference of SOD activity significant decreased (P<0.05) in 40ug/ml TP treatment group, while the difference of MDA content significant increased (P<0.05). The results of DNA ladder demonstrated that:20umol/L Aβ25-35 treatment groups had significant apoptosis ladder bands and different TP groups had different numbers of apoptotic ladder. RT-PCR was used to detect the expression of related apoptosis genes. The results showed that:control group neither express bcl-2, nor bax; Aβ25-35 group strongly express bax and only a very weak expression of bcl-2; TP treatment groups expressed both bcl-2 and bax, among which the strongest expression of bcl-2 was 20ug/ml TP group.From this research, we concluded that firstly condensed Aβ25-35 could make damage on neural stem cells by changing the cell morphology and decreaseing cell viability and the cell CAT activity. Sencondly, tea polyphenols have a protective effect on Aβ25-35-induced neural stem cells damage, through antagonizing cell oxidative stress and apoptosis, which will provide some references for using tea polyphenols to treat the central nervous system disease. |
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