| Neural stem cells(NSCs) is a kind of self-renew stem cells which can differentiate neurons, astrocytes and oligodendrocytes.The discovery of neural stem cells and their isolation and culture is important milestones in the field of stem cell research and neural bio-scientific research.They not only exist in the development stages of mammalian nervous system,but also exist in the adult mammalian nervous system.This is a challenge to the previous theory that the nervous system can not be renewable after mature,at the same time,provides a new hope for the damage repair and functional reconstruction of nervous system.Alzheimer's disease(AD),a multifaceted neurodegenerative disorder,a neurological diseases caused by brain nerve cell death,which main pathological features are as follows:the senile plaques (SP) form byβ-amyloid protein(Aβ) aggregation in the cerebral cortex and hippocampus, neurofibrillary tangles(NFT) form by Tau protein's abnormal aggregation,and nerve cells decreased in the cerebral cortex and hippocampus.High concentrations of Aβhave an neurons neurotoxicity function,Aβdeposition is an main pathogenic factor which leads to the missing of the cholinergic neurons in AD.Therefore,the deposition of Aβis the central link in the pathogenesis of AD.Aβcan cause oxidative stress,Ca2+ influx,and mitochondrial damage,then leading to nerve cell dysfunction, activating the apoptosis-related protein and factor,and ultimately launch the process of apoptosis.In addition,Aβcan also lead to neuronal apoptosis indirectly by causing brain inflammation and neurofibrillary tangles.Puerarin(Pue) is one of the major isoflavonoid compounds isolated from Radix Puerariae, which has been used in traditional Chinese medicine for centuries.Puerarin plays a main pharmacological role in the expansion of blood vessels,improvement of microcirculation, anti-oxidation,repairing of endothelial cells and inhibition apoptosis of brain damage.Research shows that:puerarin has a protective effect on injuryed nerve cells,can inhibit Aβ-induced apoptosis. Puerarin can treat vascular dementia,but there was not the clinical treatment of AD research.Effects of puerarin on the role of neural stem cells are currently not reported as well.In this study,we designed different concentrations of puerarin to intervene on the Aβ25-35 induced apoptosis of neural stem cells.In order to discuss whether puerarin has protective effects on neural stem cells or not,and study its mechanism,then providing experimental bases and references for using puerarin preventing and treating neurodegenerative diseases such as AD in clinical.In this study,we isolated neural stem cells for the experimental materials from 15-16 days of embryonic rat's brains and cultured in serum-free system,used immunocytochemistry to identify neural stem cells.Differentiated cells were identified by NSE and GFAP antibodies.Putting neural stem cells transmitted the primary culture after three passages into serum-free culture system in vitro to form neurospheres,these cell culture-hole were divided into three groups:①the control group without Aβ25-35 and puerarin②treatment group only with the Aβ25-35,added the Aβ25-35 20μmol/L condensed matter concentration into holes in the culture③Puerarin protection group,that is,at the same time adding 20μmol/L condensed matter of the Aβ25-35 and different concentrations of puerarin (adding 10-5,10-4,10-3mol/L concentration respectively).After 48 hours' culture,using inverted phase contrast microscope,Giemsa staining and DNA gel electrophoresis detecting its apoptosis,and RT-PCR detecting the apoptosis-related gene bcl-2 and bax expression levels of mRNA changes.The main results and conclusions are as follows:1.Identification of neural stem cells:After isolation and culture of NSCs from the embryonic brains, identification of NSCs with immunocytochemistry staining showed that almost all the cells were nestin-positive cells.In order to induce the NSCs differentiation,we add 10%FBS into the medium. After 4 days,almost all the cells were NSE and GFAP positive cells all can be seen by immunocytochemistry.It showed that these cells had the ability of self-renew and multidifferetiation, that is to say,these cells had basic features of NSCs.So,the cells which we isolated and cultured were NSCs.2.The morphological observation of cell apoptosis:Added DMEM medium with EGF into the third generation of the cultured cells.The result showed that Aβ25-35 injured group has the harmful effect on the cells.In contrast to the control,the shape of the cells changed with the rounding cell body,the reduction of the neurite's number,a part of cells released from the wall and some of the visible suspend cells.The different concentration of the puerarin had various protective effects on the cells. After the addition of Aβ25-35,death rate of NSCs increased strikingly with a time-dependent pattern, and the fast increasing appeared during 12-36 hours,then the increasing speed went down gradually and at the 48th hour,it amounted to 60%.After Aβ25-35 and puerarin were treated,the death rate of NSCs decreased significantly and the fast increasing was during 6-24 hours,afterwards,its death almost didn't increase.The result of Giemsa staining also showed the Aβ25-35 has injured effect on the cells and the puerarin has protective effects on the cells.3.The mRNA expression levels of apoptosis associated gene bcl-2 and bax in different groups:In control group,there was a little expression of bcl-2 and no expression of bax mRNA.In puerarin protection group,both bcl-2 and bax mRNA have expression.In 10-5mol/L puerarin,the expression of bax mRNA was more than bcl-2.10-4mol/L puerarin protection which showed the highest expression of bcl-2 and the expression of bax correspond with bcl-2.In Aβ25-35 toxic group,much more bax mRNA can be seen,whereas,bcl-2 mRNA had little expression.It shows that in vitro Aβ25-35 can inhibit the proliferation of NSCs and induce its apoptosis through activating apoptosis induction gene bax expression.The puerarin can resist the anti-apoptosis which caused by Aβ25-35 to the NSCs because puerarin can reduce the expression of anti-apoptosis gene bcl-2. |
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