| Alzheimer's disease (AD) is the most common chronic, progressive and degenerative disease in the central nervous system (CNS). AD is a progressive degenerative brain syndrome characterized by a progressive decline in memory, thinking, comprehension, calculation, language, learning capacity and judgment sufficient to impair personal activities of daily living, which result in abnormal social behaviors.β-amyloid protein (Aβ) plauques and neurofibrillary tangles are the two main features in brains of patients with AD. Plauques are dense, mostly insoluble deposits of amyloid-beta peptide and cellular material outside and around neurons. Neurofibrillary tangles are aggregates of the microtubule-associated protein tau which accumulate inside the cells. As the number of old people is growing rapidly with the development of our society, there are more and more patients with Alzheimer's diseases (AD). AD has became the fourth-leadingt causes which endangered the elderly life after the coronary heart disease, cancer and stroke. Because of the etiology and mechanism of AD is not very clear, and it can not be cured now, so it is very necessary to do some research on how to prevent and treat it.Neural stem cells are a subpolpulation of cells in the central nerve system, which have the capacity for proliferation and multiplex differentiation potential, they can differentiate into neurons, astrocytes and oligodendrocytes. When the nerve cells were damaged or dead in the central nerve system, the endogenous NSCs can act as seed cells to replace these dead cells through proliferation, differentiation and migration. But the effect of exogenous NSCs to treat AD is not satisfied. The reason is that the ability of these NSCs to survive may be weakened in the brain of patients with AD. It could be the quality problem of NSCs, or AD patients intracranial micro environmental, the changed environmental is not suitable for NSCs survival and potential play. Recently, a large number of researchs reported that the development of AD was related to the inflammation of CNS. They also reported that AD is a degenerative disease which resulted from deposition of Aβ, and deposition of A(3may also be a major factor for the progressive degeneration of neuron. It is reported that astroeytes, microglioeytes and oligodendroeytes were involved in the inflammation of CNS, these inflammatory cells can release a variety of cytokines, chemokines, alexins and their activator, which can result in non-specific inflammation, these inflammatory cell infiltration can lead to chronic inflammation and harm the micro-environment, so nerve cells derived from exogenous NSCs can not play transplantation of normal function, make the proliferation and differentiation function, or inhibition of migrating ability drops, thus the loss of nerve cells can not be replaced in time. Therefore, it is necessary for us to investgate whether imflammtory factors induced by Aβcould lead to NSCs disability, and whether activiation of a7-nAChR in microglails could improve micro-environment which can promote the ability of NSCs for proliferation and differentiation, and prevent NSCs from apoptosising.How to control the imflammtory factors induced by Aβeffectively and improve the NSCs aliving environment? It has been reported that peripheral vagus can release some neuro transmitters, these trsnsimtters can activate a7-nAChR receptors in macrophages to inhibit the synthesis and secretion of inflammatory mediators of macrophages, therefore, the peripheral inflammation can be weakened. Although there are no peripheral macrophages in the central nervous system, there are some microglials and astrocytes exist in CNS, which have the similar function of peripheral macrophages, they could be involved in the central inflammation induced by Aβ. In our early research, we have shown that these cells have a7-nAChR. Ap can cause increased secretion of IL-6, IL-1β, TNF-a, chemotactic factors MIP-1 and RANTES released by astrocytes, Aβcan also induce increased secretion of IL-6, IL-1βand TNF-a released by microglials in vitro. Activiation of a7-nAChR in microglioeytes and astrocytes could lead to reduced central inflammation induced by Aβ, and it could inhibit the activation of P38MAPK, P42/44MAPK pathway. Activiation of a7-nAChR in astrocytes could cause reduced chemotactic factors induced by Aβsignificantly and activiation of a7-nAChR in microglioeytes could lead to reduced inflammatory factor secreted by Aβobviously. Therefore, it is very important to investigate whether activation of a7-nAChR in microglioeytes could improve the NSCs micro-envrioment induced by Aβ.Based on these researchs,the aim of the present study was to investigate whether activation of a7-nAChR in microglioeytes could improve the NSCs micro-envrioment induced by Aβ, and to supply experimental foundation for further research. First of all, we successfully cultured neural stem cells and microglials from newborn rat hippocampal; secondly we establish cell inflammatory reaction model in vitro to observe the effect of Amyloid-βon proliferation, differentiation and apoptosis of neural stem cells; finally, we investigate the protection of activation microglial's a7-nAChR anginst Amyloid-P toxicity on neural stem cells. The study would help us to understand the mechanism of Alzheimer'disease and provide a new treatment target for AD.Methods1. The isolation and identification of neural stem cells (NSCs)Hippocampus were removed from Sprague-Dawley rats (within 24 hours) and digested with 0.125% trypsin at 37℃for 15 minutes. Then these cells were cultured in DMEM/F12 medium containing 20ng/ml basic fibroblast growth factor(bFGF), Epidermal growth factor(EGF) and B27; secondary passaged cells were divided into three parts as follows:BrdU incorporation assay was performed to assess the ability for self-renewal and proliferation of the NSCs; Immunofluorescence staining for Nestin was used to identify monoelonal NSCs; the third part of these cells were transferred into DMEM/F12 containing 10%FBS for 7 days, then immunofluorescence staining for glial fibrillary acidic protein (GFAP) and neuronal marker MAP-2 were used to investigate the potential multiple differentiation of NSCs.2. The isolation and identification of the microglia cellsHippocampus were removed from Sprague-Dawley rats (within 24 hours) and digested with 0.125% trypsin at 37℃for 15 minutes. These cells were cultured in DMEM/F12 medium containing 10% FBS at the density of 5×105/mL. After the cells were cultured for 7-9 days,12mmol/L lidocaine solution was added into the culture medium for 5min to purify microglia cells. Expression of CDllb/c was detected by immunofluorescence staining. Nuclei were counterstained with Hoechst33258. 3. transwell cocultureTo investigate the effect of cytokine released by microglia cells on neural stem cells. We choose a transwell coculture system. In the system, culture medium could through the semipermeable membrane aperture of 0.4μm transwell, but cells could not do this. So we put a semipermeable membrane transwell in six plastic culture plates, then we cultured neural stem cells in the upper and put microglial in the lower. Researched the secretion of microglia cells affected the neural stem cells.4. The effect of Aβon proliferation, differentiation and apoptosis of neural stem cells and the protection of activation microglial'sα7-nAChR anginst A(3 toxicity on neural stem cells.The cells were randomly divided into four groups:Control group, A(3 protein group, Coculture group and Nicotine pretreated group. BrdU assay was performed to assess the ability for self-renewal and proliferation of NSCs; expression of GFAP, MAP-2 and CHAT were detected by immunofluorescence staining to assess the multiple differentiation of NSCs, and the ratio of apoptosis was detected by Flow CytoMeter (FCM) and Fluorescence staining.5. Statistics analysisSPSS13.0 was used to analyse all of the datas. Multiple comparisons was used by ANOVA analysis (x±s), when variance not neat with Welch method. Binary comparison, while the date variance between groups together which was used LSD method, otherwise used Dunnett T3 method. The level of significance for:α= 0.05.Results1. The isolation and identification of neural stem cells (NSCs) Neural stem cells derived from hippocampus could grow quickly and they were growing as neurospheres in serum-free medium. These neurospheres were characterized by near perfect spherical shape, as well as very sharp, phase-bright outer edges. The cells in the neurospheres were positive for Nestin which is the specific marker of neural stem cells; The differentiated cells of NSCs were positive for GFAP and MAP-2. And BrdU could be combined into the nuclei. These results suggested that NSCs could proliferate and could differentiate into neurons and astrocytes.2. The isolation and identification of the microglia cellThe primary generation of mixed-cultured cells mainly included a majority of astrocytes, some microgial cells, and a minority of neurons and oligodendrocytes. After cell layers were appeared on 7-9 days, lidocaine solution was used to purify microglia cells. The cell body of microglial was flat or oval. The majority of microglia cells were positive for CDllb/c (>95%) by immunofluorescence staining.3. The effect of beta-amyloid protein (Aβ) on the ability for proliferation of neural stem cellsBrdU assay was used to detect the proliferation of neural stem cells, the ratio of proliferation in different groups were as follow:77.69%±4.45%(the Control group),52.37%±4.89%(Aβprotein group),28.67%±2.96%(Coculture group) and 39.40%±2.99%(Nicotine treatment group). There were significant difference betweet the four groups (F=576.875, P=0.000). The proliferation rate in transwell coculture group was significantly lower than Aβgroup (P<0.001). When microglial's a7-nAChR was activated anginst Aβtoxicity, the proliferation rate of NSCs increased significantly, comparing with the transwell coculture (P<0.001); however, the ratio of proliferation of NSCs in control group was significantly higher than other groups (P<0.001).4. The effect of beta-amyloid protein (A(3) on the ratio of neuron differentiated by neural stem cellsNeural stem cells were transferred in DMEM/F12 medium containing 10% FBS for 7 days. After the the neural stem cells was differentiated in DMEM/F12 medium with 10%FBS on 5 days, Aβprotein was added in control group, Aβprotein group, coculture group and Nicotine pretreatment group for 96 hours. Flow cytometry was used to detect the expression of MAP-2 in different groups. The results showed that the proportion of NSCs differentiation into neurons was 14.58% in DMEM/F12 medium containing 10% FBS; when Aβprotein was added into the medium for 96 hours, the ratio of MAP-2 positiive cells was only 10.27%, the proportion of neurons was reduced significantly (P<0.001); under the condition of microglial cells cocultrue, Aβprotein significantly reduce the ratio of neruon differentiated by NSCs, and the proportion of neurons was only 5.36%; when microglial's a7-nAChR was activated anginst Aβtoxicity, the differentiation rates of NSCs increased to 8.06%, comparing with the transwell coculture group, there was significant difference(P<0.001); However, there was significant difference between control group and other goups (F= 666.32, P= 0.000).5. The effect of beta-amyloid protein (Aβ) on the ratio of cholinergic neuron differentiated by neural stem cellsNeural stem cells could differentiated into cholinergic neuron and the expression of CHAT was detected by flow cytometry, the results showed that the positive rate of CHAT in the control group was 3.04%, it was lower than the control group(F=202.83, P=0.000); when Aβwas added in the transwell coculture for 96 hours, the rate of cholinergic neuron differentiated by the neural stem cells was less than 1%; when microglial's a7-nAChR was activated by nicotine for an hour, the ratio of cholinergic neuron was rised to 2.15%, it was higher than the transwell group (P<0.001), but still lower than the control group (P<0.001), the difference was statistically significant.6. The effect of beta-amyloid protein (AP) on apoptosis of neural stem cells and the protection of activation microglial's a7-nAChR anginst Aβtoxicity on neural stem cells.TUNNEL assay was used to investigate the ratio of apoptosis of neural stem cells. Under Fluorescence microscopy, the intensity of green fluorescence in transwell coculture group was much higher than that of Aβgroup, which suggested that there were many cells under apoptosis. There were a few cells positive for TUNNEL in nicotine treatment group, and no expression of TUNNEL was found in control group. The apoptosis rates in different groups were as follows:6.50%±0.56%(control group),25.77%±0.48%(Aβgroup),45.19%±0.52%(coculture group) and 29.17%±0.86%(nicotine treatment group). The results showed that the ratio of apoptosis induced by the transwell coculture group was significant higher than Aβgroup (P<0.001). When microglial's a7-nAChR was activated anginst Aβtoxicity, the ratio of apoptosis induced by the nicotine pretreatment group was significant lower than transwell coculture group (P<0.001), but it was higher than the control group (P<0.001). The difference was statistically significant (F=2175.97, P=0.000).Conclusions1. Neural stem cells were successfully cultured from the new born SD rats hippocampus, these cells were positive for Nestin, and these cells have the potential of multiple differentiation, which could differentiate into cells positive for astrocytes (GFAP) and neurons (MAP-2).2. Microglial cells were successfully seperated and purified with hydrochloric acid lidocaine, a majority of these cells (>95%) were positive for CD11b/c, suggested that these microglial cells could be used for coculture transwell experiment.3. Aβprotein could inhibit the ability of proliferation of NSCs and induce apoptosis of NSCs. Aβprotein could also significantly reduce the ratio of neuron differentiated by neural stem cells, especially choline neurons.4.α7-nAChR of microglia cells could be activated by nicotine, which could improve the growth microenvironment of neural stem cells and promote the ability for proliferation of neural stem cells. Activation ofα7-nAChR on microglia cells could significantly enhance the ratio of neuron differentiated by neural stem cells, especially choline neurons. |
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