Inhibitory effect of homocysteine on rat neural stem cell growth <Emphasis Type="Italic">in vitro</Emphasis> is associated with reduced protein levels and enzymatic activities of aconitase and respiratory complex III

Increased blood plasma concentration of the sulphur amino acid homocysteine (Hcy) is considered as an independent risk factor of the neurodegenerative diseases. However, the detailed molecular mechanisms by which Hcy leads to neurotoxicity have yet to be clarified. Recent research has suggested that neurotoxicity of Hcy may involve negative regulation of neural stem cell (NSC) proliferation. In the current study, primary NSCs were isolated from neonatal rat brain hippocampus and the inhibition in cell growth was observed after exposure to l50 μM and 500 μM L-Hcy. The changes in protein expression were monitored with densitometric 2D–gel electrophoresis coupled with MALDI-TOF mass spectrometry. Proteomic analysis revealed that the expression levels of two mitochondrial proteins, cytochrome bc1 complex2 (UQCRC2, the major component of electron transport chain complex III) and aconitase (an enzyme involved in the tricarboxylic acid cycle), were decreased in Hcy treatment group, compared to control group. Protein expression was further verified by Western blot, and their enzymatic activities were also down-regulated in NSCs after Hcy treatment. Restoration of aconitase and UQCRC2 protein levels using their expression vectors could partly rescue the cell viability inhibition caused by Hcy. Moreover, Hcy caused the increase in the intracellular levels of reactive oxygen species (ROS) and the decrease in ATP content, which are known to play important roles in the cellular stress response of the cell growth. Altogether, the results suggest that the decreased expression and enzymatic activities of the mitochondrial proteins may be possible causes of the overproduction of ROS and depletion of ATP. The inhibition in cell growth at the end of Hcy treatment was probably due to the changes in protein expression and mitochondrial dysfunction in vitro cultures of NSCs.     

ETOH inhibits embryonic neural stem/precursor cell proliferation via PLD signaling

While a mother’s excessive alcohol consumption during pregnancy is known to have adverse effects on fetal neural development, little is known about the underlying mechanism of these effects. In order to investigate these mechanisms, we investigated the toxic effect of ethanol (ETOH) on neural stem/precursor cell (NSC) proliferation. In cultures of NSCs, phospholipase D (PLD) is activated following stimulation with epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2). Exposure of NSCs to ETOH suppresses cell proliferation, while it has no effect on cell death. Phosphatidic acid (PA), which is a signaling messenger produced by PLD, reverses ETOH inhibition of NSC proliferation. Blocking the PLD signal by 1-butanol suppresses the proliferation. ETOH-induced suppression of NSC proliferation and the protective effect of PA for ETOH-induced suppression are mediated through extracellular signal-regulated kinase signaling. These results indicate that exposure to ETOH impairs NSC proliferation by altering the PLD signaling pathway.     

Effects of Sevoflurane on Self-Renewal Capacity and Differentiation of Cultured Neural Stem Cells

Sevoflurane anesthesia in infant rats can result in long-term cognitive impairment, possibly by inhibiting neurogenesis. The hippocampus is critical for memory consolidation and is one of only two mammalian brain regions where neural stem cells (NSCs) are renewed continuously throughout life. To elucidate the pathogenesis of sevoflurane-induced cognitive dysfunction, we measured the effects of clinical sevoflurane doses on the survival, proliferation, and differentiation of hippocampal NSCs. Neural stem cells were isolated from Sprague–Dawley rat embryos, expanded in vitro, and exposed to sevoflurane at 0.5, 1, or 1.5 minimal alveolar concentration (MAC) for 1 or 6 h. Two days after treatment, cell viability, cytotoxicity, and apoptosis rate were estimated by WST-1 assay, lactate dehydrogenase (LDH) activity, and TdT-mediated dUTP-biotin nick end labeling (TUNEL), respectively, while proliferation rate was assessed by 5-ethynyl-2′-deoxyuridine (BrdU) incorporation and Ki67 staining. Differentiation was assayed 7 days after treatment by immunocytochemistry and Western blots of neuron and glial markers. The phosphorylation level of p44/42 extracellular regulated kinases (ERK1/2) was measured in the proliferation and differentiation phases respectively. Sevoflurane at 1 MAC or 1.5 MAC for 1 h increased viable cell number whereas a 6 h exposure at these same concentrations suppressed proliferation and promoted apoptotic death (P < 0.01). Sevoflurane had no effect on NSC differentiation, and a sub-clinical concentration (0.5 MAC) altered neither proliferation nor viability. The phosphorylation level of ERK1/2 increased after 1 h of 1 MAC or 1.5 MAC of sevoflurane exposure in the proliferation phase, but not in the differentiation phase. Brief (1 h) exposure to sevoflurane at clinical concentrations enhanced proliferation of cultured NSCs possibly mediated by ERK1/2, but a 6 h exposure suppressed proliferation and induced apoptosis. Prolonged sevoflurane exposure may decrease the self-renewal capacity of hippocampal NSCs, resulting in cognitive deficits.     

Effect of All-Trans Retinoic Acid on the Differentiation of U87 Glioma Stem/Progenitor Cells

GSPCs (glioma stem/progenitor cells) were isolated from U87 glioma cell lines by serum-free neural stem cell medium. Four concentrations (1, 2, 4, and 8 μmol/L) of ATRA (all-trans retinoic acid) were used to induce the differentiation of GSPCs in the medium with or without growth factors. The effect of ATRA on the differentiation of GSPCs was analyzed by flow cytometry, real-time-PCR, and immunofluorescence. The differentiation of GSPCs could be induced by 1 or 2 μmol/L ATRA when GSPCs were cultured in growth factor-free medium. The detection of real-time-PCR showed that the level of GFAP (glial fibrillary acidic protein) mRNA of differentiated GSPCs in the growth factor-free medium containing 1 μmol/L ATRA group was significantly higher than that in the control group, and there was no significant difference in the level of TUBB-3 mRNA between the two groups. The GSPCs suffered apoptosis in the growth factor-free medium containing 4 or 8 μmol/L ATRA. The differentiation of GSPCs could not be induced by ATRA when GSPCs were cultured in the medium containing growth factors. The percentage of cells in G0/G1 phase was 84.26 ± 2.24 %, and the percentage of apoptosis was 18.95 ± 2.53 % in experimental groups which was similar to those in the control group. In conclusion, ATRA has certain capacity to induce differentiation of GSPCs, while its effective concentration should be controlled strictly. The differentiation of GSPCs induced by ATRA cannot antagonize the formidable differential inhibition of epidermal growth factor and basic fibroblast growth factor.     

Melatonin Increases Oligodendrocyte Differentiation in Cultured Neural Stem Cells

Neural stem cell (NSC) culture is a remarkable tool to investigate the potential therapeutic benefits of drugs in neurological diseases. The purpose of this study was to determine the effect of melatonin on proliferation and differentiation of NSCs in vitro. NSCs were isolated and expanded from mouse embryonic E14 cortex, and the effect of various concentrations of melatonin (0.05, 0.1, 0.5, 1, 5 and 10 μM) on NSC proliferation was assessed by MTT and neurosphere assay. Results showed that melatonin significantly increased NSC viability and NSC proliferation in a dose-dependent manner, in comparison to controls. Similarly, neurosphere formation frequency and cell counts increased significantly with increasing melatonin concentrations and reached its peak at 0.5 μM, in comparison to controls. Moreover, NSCs treated with either low (0.05 µM) or high concentrations (5 µM) of melatonin showed that the mean percentage of glial fibrillary acidic protein (GFAP) positive cells were not significantly different in PDGF or melatonin at 5 μM, in comparison to controls. However, low melatonin concentrations (0.05 µM) showed a slight significant increase in comparison to controls and PDGF. On the other hand, both concentrations of melatonin treatment significantly increased the percentage of myelin basic protein (MBP) positive cells (oligodendrocytes), in comparison to controls and to PDGF. Our results demonstrated, for the first time, that melatonin increased oligodendrocyte differentiation from NSCs. These results suggest that melatonin might have a potential therapeutic effect for some neurological diseases that involve oligodendrocyte and myelin pathologies.     

Low expression of ERK signaling pathway affecting proliferation,cell cycle arrest and apoptosis of human gastric HGC-27 cells line

This study was carried out for the first time to examine the potential role and the underlying mechanisms of Lycopene in the gastric cancer HGC-27 cells. HGC-27 cells were seeded onto heat-sterilized coverslips in six-well plates and exposed to Lycopene (5, 10, 20, 30 and 40 μmol/L) for periods of 72 h at 37 °C. Results showed that Lycopene (5, 10, 20, 30 and 40 μmol/L) dose-dependently increased NBT positive rate and decreased lactate dehydrogenase activity in HGC-27 cells. In addition, Lycopene (5, 10, 20, 30 and 40 μmol/L) inhibited proliferation and induced G0–G1 phase cell cycle arrest in HGC-27 cells. Western blot and FQRT-PCR analysis showed that Lycopene decreased pERK and extracellular signal-regulated kinase (ERK) protein and mRNA expression in a dose-dependent manner. These findings demonstrate that Lycopene inhibited gastric cancer HGC-27 cells growth and stimulated its apoptosis via the suppressing ERK signaling pathway.     

Purinergic signaling modulates the cerebral inflammatory response in experimentally infected fish with <Emphasis Type="Italic">Streptococcus agalactiae</Emphasis>: an attempt to improve the immune response

Epigallocatechin gallate (EGCG), a bioactive ingredient of green tea, plays a protective role in the cardiovascular system. Homocysteine (Hcy) is a major risk factor for chronic kidney disease and cardiovascular disease. The present study aimed to investigate the role of EGCG in Hcy-induced proliferation of vascular smooth muscle cells (VSMCs) and its underlying mechanism. We also explored the roles of rennin-angiotensin system (RAS), extracellular signal-regulated kinases (ERK1/2), and p38 mitogen-activated protein kinase (p38 MAPK) in this process. Human aortic smooth muscle cells (HASMCs) were treated with different drugs for different periods. The proliferation rate of HASMCs was detected using the CCK-8 and BrdU labeling assays. The Western blot assay was used to determine the expression levels of angiotensin II type 1 receptor (AT-1R), ERK1/2, and p38 MAPK. Compared with the control group, the HASMCs treated with Hcy at different doses (100, 200, 500, and 1000 µM) showed significantly increased proliferation. Hcy increased the expression of AT-1R, whereas EGCG decreased the protein expression of AT-1R. Furthermore, we found that Hcy-induced expression of p-ERK1/2 and p-p38MAPK was dependent on AT-1R. Compared with Hcy (500 µM)-treated cells, EGCG (20 µM)-treated cells showed decreased proliferation as well as expression of AT-1R, p-ERK1/2, and p-p38MAPK. In addition, HASMC proliferation was suppressed by the addition of an AT-1R blocker (olmesartan), an ERK1/2 inhibitor (PD98059), and a p38MAPK inhibitor (SB202190). EGCG can inhibit AT-1R and affect ERK1/2 and p38MAPK signaling pathways, resulting in the decrease of VSMC proliferation induced by Hcy.     

胎鼠脊髓源性神经干细胞分离培养与鉴定

目的:研究胎鼠的脊髓源性神经干细胞的分离培养方法并观察其增殖和分化能力。方法:利用显微操作技术分离获得胎鼠脊髓组织、无血清培养技术和酶消化法结合机械法传代培养神经干细胞、免疫细胞化学方法鉴定神经干细胞和分化情况。结果:建立了胎鼠脊髓源性神经干细胞的分离、培养和鉴定的方法,观察到了脊髓源性神经干细胞具有较强的增殖能力,在添加有5ng／mlEGF和5ng／mlbFGF的无血清培养液中可贴壁分化为神经元、少突细胞和星形胶质细胞。结论:在体外培养条件下分离培养的胎鼠脊髓源性神经干细胞具有干细胞的特性即较强的增殖能力和多向分化潜能。     

Effect and mechanism of mGluR6 on the biological function of rat embryonic neural stem cells

Here, we investigated the effects and molecular mechanisms of metabotropic glutamate receptor 6 (mGluR6) on rat embryonic neural stem cells (NSCs). Overexpression of mGluR6 significantly promoted the proliferation of NSCs and increased the diameter of neutrospheres after treatment for 24 h, 48 h and 72 h. Overexpression of mGluR6 promoted G1 to S phase transition, with significantly decreased cell ratio in G1/G0 phase but significantly increased cell ratio in S phase. Additionally, mGluR6 overexpression for 48 h decreased the early and late apoptosis significantly. Moreover, overexpression of mGluR6 significantly increased the expression of p-ERK1/2, Cyclin D1 and CDK2, while the expression of p-p38 was significantly decreased. On the contrary, these effects of mGluR6 overexpression were reversed by mGluR6 knockdown. In conclusion, mGluR6 promotes the proliferation of NSCs by activation of ERK1/2-Cyclin D1/CDK2 signaling pathway and inhibits the apoptosis of NSCs by blockage of the p38 MAPK signaling pathway.     

DNA methyltransferase mediates dose-dependent stimulation of neural stem cell proliferation by folate

The proliferative response of neural stem cells (NSCs) to folate may play a critical role in the development, function and repair of the central nervous system. It is important to determine the dose-dependent effects of folate in NSC cultures that are potential sources of transplantable cells for therapies for neurodegenerative diseases. To determine the optimal concentration and mechanism of action of folate for stimulation of NSC proliferation in vitro, NSCs were exposed to folic acid or 5-methyltetrahydrofolate (5-MTHF) (0–200 μmol/L) for 24, 48 or 72 h. Immunocytochemistry and methyl thiazolyl tetrazolium assay showed that the optimal concentration of folic acid for NSC proliferation was 20–40 μmol/L. Stimulation of NSC proliferation by folic acid was associated with DNA methyltransferase (DNMT) activation and was attenuated by the DNMT inhibitor zebularine, which implies that folate dose-dependently stimulates NSC proliferation through a DNMT-dependent mechanism. Based on these new findings and previously published evidence, we have identified a mechanism by which folate stimulates NSC growth.     

Homocysteine induces procoagulant activity of red blood cells via phosphatidylserine exposure and microparticles generation

Increased homocysteine (Hcy) levels in plasma correlate with the risk of thromboic events. Red blood cells (RBCs), the most abundant blood cells in circulation, also play an active role in the process of thrombus formation. However, the effect of Hcy on procoagulant activity (PCA) of RBCs is unclear. In the present study, RBCs from healthy adults were treated with Hcy (8, 20, 80, 200, 800 μmol/L) for 24 h. Phosphatidylserine (PS) exposure of RBCs and red blood cell-derived microparticles (RMPs) release were detected using Alexa Fluor 488-lactadherin. PCA was assessed by coagulation time and purified clotting complexes testes. We found that Hcy treatment dose dependently enhanced PS exposure and consequent PCA of RBCs. Hcy also elevated the formation of procoagulant RMPs, with statistical significance at 800 μmol/L of Hcy. Moreover, 128 nmol/L lactadherin inhibited about 90 % PCA of RBCs and RMPs. Our data suggest that PS exposure and RMPs shedding are key sources for Hcy-induced PCA of RBCs. Lactadherin could be used to modulate the anticoagulant and procoagulant balance in this process.     

Role of miRNA-146?in proliferation and differentiation of mouse neural stem cells

Neural stem cells (NSCs) have been defined as neural cells with the potential to self-renew and eventually generate all cell types of the nervous system. NSCs serve as an ideal cell type for nervous system repair. In the present study, miR-146 overexpression and predicted target (notch 1) were used to study proliferation and differentiation of mouse NSCs. shRNA were used to demonstrate the function of Notch 1 in proliferation of mouse NSCs and luciferase reporter assay was used to assess and confirm the binding sequence of 3′-UTR between Notch 1 and miR-146. Results showed that miR-146 overexpression and knockdown of notch 1 inhibited proliferation of mouse NSCs under serum-free cultural conditions and promoted spontaneous differentiation of mouse NSCs under contained serum cultural conditions respectively. Mouse NSCs spontaneously underwent differentiation into neurogenic cells with contained serum medium. However, when miR-146 was overexpressed, differentiation efficiency of glial cells from NSCs was increased, suggesting that Notch1 promoted NSC proliferation and repressed spontaneous differentiation of NSC in serum-free medium. In conclusion, our results demonstrate that miR-146 promoted spontaneous differentiation of NSCs, and this mechanism was influenced by miR-146, as well as its target (notch 1) and downstream gene.     

细胞外信号调节激酶在小鼠神经干细胞增殖中的作用

本文旨在探讨细胞外信号调节激酶(extracellular signal-regulated kinase 1/2, ERK1/2)对小鼠神经干细胞增殖的影响.分离E14.5小鼠皮层神经干细胞,通过Western blot检测神经干细胞增殖过程中磷酸化ERK1/2的表达情况,以及不同浓度PD98059处理对神经干细胞ERK1/2磷酸化及神经球形成的影响,并用CCK-8法检测PD98059对神经干细胞增殖的影响.结果显示:ERK1/2在体外培养的神经下细胞增殖过程中被激活;PD98059显著抑制ERK1/2磷酸化及神经干细胞的成球率,且存在剂量效应依赖关系;加入PD98059后神经干细胞的生长被抑制.以上结果表明,ERK1/2在小鼠神经干细胞增殖中具有重要的作用,阻断ERK1/2信号通路后可抑制神经干细胞的增殖.     

Low-Dose Homocystine Enhances Proliferation and Migration of Bv2 Microglia Cells

Homocysteine (Hcy) is a non-essential amino acid that is derived from the breakdown of dietary methionine. Hyperhomocysteinemia (HHcy) is an independent risk factor for a variety of chronic diseases, especially neurodegenerative conditions. To better understand the role of HHcy in the pathogenesis of neurodegenerative disorders, we investigated the effect of Hcy on the proliferation and activation of microglia Bv2 cells. Cells were treated with six different Hcy concentrations: 0, 50, 100, 300, 500, and 1000 µM for different time periods (8, 12, 16, 24, and 48 h). The morphology of Bv2 cells was observed, and cell activity and proliferation were detected. Cell migration and secretion of pro-inflammatory cytokines were detected by the scratch wound assay, the transwell assay, and ELISA, respectively. The effect of Hcy on Bv2 proliferation occurred earlier (<24 h, especially 16 h) after treatment with concentrations between 100 and 300 μM, and there was no cytotoxicity to Bv2 cells. Meanwhile, functional assays suggested that Hcy not only promoted Bv2 secretion of the pro-inflammatory cytokines IL-1β, TNF-α, and IL-6, but also enhanced Bv2 migration and invasion, with 100 μM being the most effective concentration. In summary, Bv2 proliferation and activation can be promoted by short-term treatment with low-dose Hcy.     

Wnt and Shh signals regulate neural stem cell proliferation and differentiation in the optic tectum of adult zebrafish

Adult neurogenesis occurs more commonly in teleosts, represented by zebrafish, than in mammals. Zebrafish is therefore considered a suitable model to study adult neurogenesis, for which the regulatory molecular mechanisms remain little known. Our previous study revealed that neuroepithelial‐like neural stem cells (NSCs) are located at the edge of the dorsomedial region. We also showed that Notch signaling inhibits NSC proliferation in this region. In the present study, we reported the expression of Wnt and Shh signaling components in this region of the optic tectum. Moreover, inhibitors of Wnt and Shh signaling suppressed NSC proliferation, suggesting that these pathways promote NSC proliferation. Shh is particularly required for maintaining Sox2‐positive NSCs. Our experimental data also indicate the involvement of these signaling pathways in neural differentiation from NSCs. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1206–1220, 2017     

Upregulation of Flk-1 by bFGF via the ERK pathway is essential for VEGF-mediated promotion of neural stem cell proliferation

Neural stem cells （NSCs） constitute the cellular basis for embryonic brain development and neurogenesis. The process is regulated by NSC niche including neighbor cells such as vascular and glial cells. Since both vascular and glial cells secrete vascular endothelial growth factor （VEGF） and basic fibroblast growth factor （bFGF）, we assessed the effect of VEGF and bFGF on NSC proliferation using nearly homogeneous NSCs that were differentiated from mouse embryonic stem cells. VEGF alone did not have any significant effect. When bFGF was added, however, VEGF stimulated NSC proliferation in a dose-dependent manner, and this stimulation was inhibited by ZM323881, a VEGF receptor （Flk-1）- specific inhibitor. Interestingly, ZM323881 also inhibited cell proliferation in the absence of exogenous VEGF, suggesting that VEGF autocrine plays a role in the proliferation of NSCs. The stimulatory effect of VEGF on NSC proliferation depends on bFGF, which is likely due to the fact that expression of Flk-1 was upregulated by bFGF via phosphorylation of ERK1/2. Collectively, this study may provide insight into the mechanisms by which microenvironmental niche signals regulate NSCs.     

Peroxisome proliferator-activated receptor gamma-mediated regulation of neural stem cell proliferation and differentiation

Peroxisome proliferator-activated receptor gamma (PPARgamma) plays an important role in insulin sensitivity, tissue homeostasis, and regulating cellular functions. We found high-level expression of PPARgamma in embryo mouse brain and neural stem cells (NSCs), in contrast to extremely low levels in adult mouse brain. Here, we show that PPARgamma mediates the proliferation and differentiation of murine NSCs via up-regulation of the epidermal growth factor receptor and activation of the ERK pathway. Cell growth rates of NSCs prepared from heterozygous PPARgamma-deficient mouse brains, PPARgamma-RNA-silenced NSCs, and PPARgamma dominant-negative NSCs were significantly decreased compared with those of wild-type NSCs. Physiological concentrations of PPARgamma agonists, rosiglitazone and pioglitazone, stimulated NSC growth, whereas antagonists caused cell death in a concentration-dependent manner via activation of the caspase cascade. The stimulation of cell growth by PPARgamma was associated with a rapid activation of the ERK pathway by phosphorylation and up-regulation of epidermal growth factor receptor and cyclin B protein levels. In contrast, activation of PPARgamma by agonists inhibited the differentiation of NSCs into neurons. The inhibition of differentiation was associated with an activation of STAT3. These data indicate that PPARgamma regulates the development of the central nervous system during early embryogenesis via control of NSC proliferation.     

溶血磷脂酸促进离体大鼠胚胎神经干细胞的增殖及其向胆碱能神经元分化

溶血磷脂酸（1ysophosphatidic acid,LPA）是一种细胞外磷脂信号。本研究用[^3H]-胸腺嘧啶掺入法、免疫细胞化学和Western blot等技术,观察了LPA对体外培养的大鼠胚胎神经干细胞（neural stem cells,NSCs）的增殖以及向MAF2标记的一般神经元和ChAT标记的胆碱能神经元的分化的影响。结果显示：（1）在特殊的无血清培养基中加入低浓度的LPA（0．01-1．0μmol/L）后,NSCs对【^3H】-胸腺嘧啶的摄入呈剂量依赖性增加,表明LPA对NSCs有显著的促增殖作用;（2）在培养基中加入胎牛血清以诱导NSCs的分化,发现低浓度的LPA增加MAF2阳性和ChAT阳性神经元的比例,0．1μmol／L LPA引起的增加达到峰值;（3）Western blot分析显示LPA促进了MAP2和ChAT的表达;（4）在诱导NSCs出现分化早期,用倒置显微镜观察到低浓度的LPA明显促进细胞突起的生长和细胞的迁移。以上结果表明,低浓度LPA在一定范围内可以促进NSCs的增殖、并分化为一般的MAP2阳性神经元和特殊的胆碱能神经元,而且LPA可以促进在分化早期出现的神经元或神经胶质细胞前体细胞的迁移和突起生长。     

Upregulation of FIk-1 by bFGF via the ERK pathway is essential for VEGF-mediated promotion of neural stem cell proliferation

Neural stem cells (NSCs) constitute the cellular basis for embryonic brain development and neurogenesis.The processis regulated by NSC niche including neighbor cells such as vascular and glial cells.Since both vascular and glial cellssecrete vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF),we assessed the effect ofVEGF and bFGF on NSC proliferation using nearly homogeneous NSCs that were differentiated from mouse embryonicstem cells.VEGF alone did not have any significant effect.When bFGF was added,however,VEGF stimulated NSCproliferation in a dose-dependent manner,and this stimulation was inhibited by ZM323881,a VEGF receptor (Flk-1)-specific inhibitor.Interestingly,ZM323881 also inhibited cell proliferation in the absence of exogenous VEGF,suggestingthat VEGF autocrine plays a role in the proliferation of NSCs.The stimulatory effect of VEGF on NSC proliferationdepends on bFGF,which is likely due to the fact that expression of Flk-1 was upregulated by bFGF via phosphoryla-tion of ERK1/2.Collectively,this study may provide insight into the mechanisms by which mieroenvironmental nichesignals regulate NSCs.     

光遗传技术通过Wnt/β-Catenin通路对新生神经元的影响*

目的:探讨Wnt/β-catenin信号通路光遗传技术在促进新生神经元成熟中的作用。方法:从胎鼠大脑皮层中提取神经干细胞,用携带DCX-ChR2-EGFP基因的慢病毒感染神经干细胞,观察神经干细胞分化为新生神经元后DCX的表达。实验细胞分为3组(n=9):对照组、NSCs+EGFP和NSCs+ChR2组。其中对照组为正常培养的NSCs(NSCs组);NSCs+EGFP组为携带DCX-EGFP基因慢病毒感染神经干细胞组;NSCs+ChR2组为携带DCX-ChR2-EGFP基因慢病毒感染神经干细胞组。病毒感染后48 h后连续3 d行470 nm蓝激光照射,然后检测各组NeuN⁺阳性细胞(成熟神经元标志物)的密度和NeuN⁺/Hoechst比值情况;Western blot检测各组成熟神经元相关蛋白MAP2、NeuN、Neurog2、NeuroD1和GluR2蛋白表达水平和Wnt/β-catenin通道相关蛋白TCF4和β-catenin蛋白的表达水平。用L-型钙通道阻断剂100 μmol/L维拉帕米或50 μg/ml的β-catenin抑制剂Dkk1处理NSCs+ChR2组细胞,然后行Western blot检测各组MAP2、NeuN、Neurog2、NeuroD1和GluR2蛋白表达水平。结果:连续3 d 470 nm蓝激光照射后,NSCs+ChR2组中NeuN⁺阳性细胞密度(成熟细胞)和NeuN⁺/Hoechst明显高于NSCs组和NSCs+EGFP组(P均＜0.05);Western blot检测的MAP2、NeuN、Neurog2、NeuroD1、GluR2蛋白及Wnt/β-catenin通路相关蛋白β-catenin、TCF4表达水平均明显高于NSCs组和NSCs+EGFP组(P均＜0.01);L-型钙通道阻断剂维拉帕米或β-catenin抑制剂Dkk1处理NSCs+ChR2组细胞后MAP2、Neurog2、NeuroD1和GluR2蛋白表达水平明显下降(P均＜ 0.01),NeuN表达水平也下降(P＜0.05)。证明ChR2通道蛋白开放产生阳离子内流促进新生神经元成熟,是通过Wnt/β-catenin信号通路实现的。结论:光遗传学方法通过Wnt/β-catenin信号通路促进新生神经元成熟。