BACKGROUND:Although astrocytes are known to contribute to Alzheimer's disease (AD) progression, their dynamic molecular alterations remain poorly characterized, particularly in early stages of the disease. METHODS:We performed multi-omics profiling (transcriptomics, proteomics, spatial metabolomics) of astrocytes from APP/PS1 and WT mice to characterize dynamic changes during AD progression. To assess similar changes in early human AD, we analyzed single-nucleus RNA sequencing data from human samples. RESULTS:Transcriptomic analysis of astrocytes from APP/PS1 and WT mice at five time points (2, 4, 6, 9, and 12 months of age) showed notable gene expression differences at 6 months, with reduced activity in fatty acid metabolism pathways (e.g., PPAR signaling, biosynthesis of unsaturated fatty acids). An astrocyte-specific metabolic model confirmed these disruptions. Proteomic analysis corroborated this by showing decreased activity in pathways like butanoate metabolism and PPAR signaling. Spatial metabolomics of brain slices from APP/PS1 and WT mice highlighted fatty acid enrichment in the hippocampus and cortex, alongside differential metabolites specific to the AD mouse model. Single-cell RNA sequencing analysis of human brain samples further showed fatty acid metabolism abnormalities in astrocytes from early AD cases versus controls, emphasizing its role in AD progression. CONCLUSION:Our study identified abnormal fatty acid metabolism as an early feature of astrocytes in AD, suggesting an association between dysregulated fatty acid metabolism and disease progression.

Alzheimer's Disease (AD) is a neurodegenerative condition characterized by the accumulation and deposition of amyloid-β (Aβ) aggregates in the brain. Despite a wealth of research on the toxicity of Aβ and its role in synaptic damage, the mechanisms facilitating Aβ clearance are not yet fully understood. However, microglia, the primary immune cells of the central nervous system, are known to maintain homeostasis through the phagocytic clearance of protein aggregates and cellular debris. In this study, RNA sequencing analysis and live cell functional screens are employed to uncover microglial genetic modifiers related to AD. Lyzl4 is identified, which encodes a c-type lysozyme-like enzyme primarily localized to microglial lysosomes, as a gene significantly upregulated in AD microglia with aging and propose that Lyzl4 upregulation acts as a positive regulator of Aβ clearance. Furthermore, it is found that Lyzl4 overexpression boosts Aβ clearance both in vitro and in vivo, underscoring its potential for mitigating Aβ burden. These novel insights position Lyzl4 as a promising therapeutic target for Alzheimer's disease, paving the way for further exploration into potential AD treatments.

BackgroundInterleukin-17 (IL-17) family cytokines play critical roles in inflammation and pathogen resistance. Inflammation in the central nervous system, denoted as neuroinflammation, promotes the onset and progression of Alzheimer's disease (AD). Previous studies showed that IL-17A neutralizing antibody treatment alleviated Amyloid beta (A beta) burden in rodent models of AD, while overexpression of IL-17A in mouse lateral ventricles rescued part of the AD pathology. However, the involvement of IL-17 in AD and its mechanism of action remain largely unknown.MethodsTo investigate the role of IL-17 in AD, we crossed mice lacking the common receptor of IL-17 signaling (IL-17RA knockout mice) to the APP/PS1 mouse model of AD. We then analyzed the composition of immune cells and cytokines/chemokines during different phases of AD pathology, and interrogated the underlying mechanism by which IL-17 may regulate immune cell infiltration into AD brains.ResultsAblation of IL-17RA in APP/PS1 mice decreased infiltration of CD8+ T cells and myeloid cells to mouse brain. IL-17 was able to promote the production of myeloid- and T cell-attracting chemokines CXCL1 and CXCL9/10 in primary glial cells. We also observed that IL-17 is upregulated in the late stage of AD development, and ectopic expression of IL-17 via adenoviral infection to the cortex trended towards worsened cognition in APP/PS1 mice, suggesting a pathogenic role of excessive IL-17 in AD.ConclusionOur data show that IL-17 signaling promotes neuroinflammation in AD by accelerating the infiltration of CD8+ T lymphocytes and Gr1+ CD11b+ myeloid cells.

ANKRD11 gene encodes for the large nuclear protein essential for multiple system development including the nervous system. However, the molecular basis for the proper nuclear localization of ANKRD11 has not yet been elucidated. In this study, we have identified a functional bipartite nuclear localization signal (bNLS) between residues 53 and 87 of ANKRD11. Using biochemical approaches, we discovered two major binding sites in this bipartite NLS for Importin α1. Through site-directed mutagenesis and functional analysis, we further found that this bipartite NLS is sufficient for nuclear import of overexpressing GFP in HeLa cells and necessary for nuclear localization of ANKRD11. Importantly, our study provides a possible pathogenic mechanism for certain clinical variants located within the bipartite nuclear localization signal of ANKRD11.

BackgroundPeriodontitis has become an increasingly important public health issue, coupled with a high economic burden for prevention and treatment. Exposure to essential trace heavy metals has been associated with various diseases; however, the relationships between essential trace heavy metals and periodontitis remain inconclusive.ObjectivesTo investigate the association between essential trace heavy metals in tap water and periodontitis in a nationally representative sample in China.MethodsWe conducted a nationwide study including 1348 participants from the Fourth National Oral Health Survey in the 2015-2016 period. The trace heavy metals concentration was measured in the local pipeline terminal tap water. Periodontitis was diagnosed according to the classification scheme proposed at the 2018 world workshop on the classification of periodontal and peri-implant diseases and conditions. We used weighted multivariable logistic regression to estimate the association between essential trace heavy metals and the risk of periodontitis. We additionally used spline analysis to explore the possible nonlinear dose-response associations.ResultsPeriodontitis patients were exposed to higher concentrations of essential trace heavy metals. In adjusted models, for 1 SD increase in the concentration of iron, manganese, and copper in tap water, the risk of periodontitis increased by 30% (OR: 1.30, 95%CI: 1.12-1.50), 20% (OR: 1.20, 95%CI: 1.03-1.41), and 20% (OR: 1.20, 95%CI: 1.04-1.39), respectively. Stratified analyses demonstrated that the associations between essential trace heavy metals and periodontitis were higher in females, elders, and rural residents. Spline analysis revealed nonlinear exposure-response relationships between periodontitis and exposure to iron, manganese, and copper in tap water.ConclusionsExposures to essential trace heavy metals in drinking water were associated with greater odds of periodontitis. Given the growing burden of periodontitis, our study sheds light on tailored public health policies for improving drinking water standards to alleviate periodontitis impairment.

CASK is a unique scaffold protein in the synapse system. It links numerous proteins to the pre- or post-synaptic region and is critical to the homeostasis of synaptic vesicles. The N-terminus of CASK is a calcium/calmodulin-dependent protein kinase (CAMK) domain, which has diverse functions and interacts with downstream proteins to form a scaffold platform. Caskin1 is one of the brain-specific adaptor proteins of CASK. Previous studies showed that CASK_CAMK domain interacts with Caskin1 CID domain with relatively low affinity. In this study, we re-visit this interaction by remapping the interaction boundary and solving their complex structure. Based on the structure, we systematically compared the interactions between CASK_CAMK and other binding partners. Our results showed that CAMK domain occupies the CID peptide by using its C-lobe groove (between the α1 and α2) and there is a highly conserved signature motif (ζ-x-ψ-W-ψ-x-R) in the CID domain, where ζ is acidic side chain containing residues, x is any amino acid residue, ψ is hydrophobic residues, W is for tryptophan, and R is arginine. These findings allowed us to identify several new potential cytoplasmic binding partners for CASK_CAMK.

Background Increasing evidence indicates that the aberrant expression of circular RNAs (circRNAs) is involved in the pathogenesis and progression of lung adenocarcinoma (LUAC). However, the function and molecular mechanisms of hsa_circ_0002483 (circ_0002483) in LUAC remain unclear. Methods The association between circ_0002483 expression and clinicopathological characteristics and prognosis in patients with LUAC was analyzed by fluorescence in situ hybridization. The functional experiments such as CCK-8, colony formation and Transwell assays and a subcutaneous tumor model were conducted to determine the role of circ_0002483 in LUAC cells. The specific binding between circ_0002483 and miR-125a-3p was validated by RNA immunoprecipitation, luciferase gene report and qRT-PCR assays. The effects of circ_0002483 on miR-125a-3p-mediated C-C motif chemokine ligand 4 (CCL4)-CCR5 axis were assessed by Western blot analysis. Results We found that circ_0002483 was upregulated in LUAC tissue samples and associated with Tumor Node Metastasis (TNM) stage and poor survival in patients with LUAC. Knockdown of circ_0002483 inhibited proliferation, colony formation and invasion of A549 and PC9 cells in vitro, whereas overexpression of circ_0002483 harbored the opposite effects. Furthermore, circ_0002483 sponged miR-125a-3p and negatively regulated its expression. CCL4 was identified as a direct target of miR-125a-3p. The rescue experiments showed that miR-125a-3p mimics reversed the tumor-promoting effects of circ_0002483 by targeting CCL4-CCR5 axis in A549 and PC9 cells. In addition, the in vivo experiment further validated that knockdown of circ_0002483 repressed tumor growth. Conclusions Our findings demonstrated that circ_0002483 could act as a sponge of miR-125a-3p to upregulate CCL4-CCR5 axis, contributing to the tumorigenesis of LUAC, and represent a potential therapeutic target for LUAC.

目的:探讨Pax3的过表达对Neuro-2a细胞中转录本的表达影响,初步分析Pax3对Neuro-2a细胞可能的转录调控作用.方法:反复冻融裂解法获取Pax3过表达腺病毒后将神经瘤母细胞系Neuro-2a传代培养,而后将Pax3过表达腺病毒和传代培养后的Neuro-2a细胞加入到同一培养皿中,蛋白质免疫印迹(Western blot)检测过表达Pax3蛋白的Neuro-2a细胞(Pax3过表达组)和对照组(NC组)Neuro-2a细胞的Pax3蛋白表达水平,实时荧光定量PCR(qRT-PCR)法检测Pax3过表达组和NC组Neuro-2a细胞的Pax3mRNA水平,Trizol法提取Pax3过表达组和NC组Neuro-2a细胞的总RNA,然后进行全转录本测序,最后将选出的有差异性的基因使用实时荧光定量PCR(qRT-PCR)验证.结果:与NC组相比,Pax3过表达组的Pax3蛋白和Pax3mRNA表达水平明显升高(P＜0.05);Pax3过表达组中发现了1045个基因表达上调,1313个基因表达下调.通过qRT-PCR验证发现在Pax3过表达组中Nppb和Chrna5表达水平上升(P＜0.05),Arhgap5、Rock1、Rif1、Brca2、Prkg2和Stag2表达水平下降(P＜0.05).结论:Pax3过表达腺病毒感染Neuro-2a细胞后,其蛋白和mRNA表达水平均升高,Rock1、Rif1和Stag2可能作为Pax3的下游靶点参与调控Neuro-2a细胞周期和干细胞特性.

Lung cancer is a most common cancer worldwide. Tumor-associated macrophage (TAM) is known a key effector cell in tumor microenvironment. Meanwhile, STAT6 is crucial to cancer development. We aimed to determine the interaction between STAT6 and TAMs in lung cancer. In this work, firstly, we established mouse model of lung cancer. Then, immunofluorescence was performed to determine STAT6 and CD206 level in lung cancer tissue and adjacent normal tissues as well as model mice. RT-qPCR was applied to detect differentiation of macrophage and determine related gene expression. After treatment of siRNA of STAT6 or STAT6 inhibitor (AS1517499), Transwell assay and MTT were used to determine cell proliferation and migration. STAT6 was upregulated in lung cancer tissues while arginase was more active in M2 macrophage rather than M1 macrophage. Transfection of si-STAT6 not only decreased differentiation in M2 macrophage but also inhibited proliferative, migratory and invasive ability of cancer cells while AS1517499 led to reduced tumor growth. STAT6 inhibition caused decreased expression of M2 macrophages. Similarly, intratumoral T cell markers showed that CD8+T cell gene expression and CD4-mediated T cell marker FoxP3 was increased slightly. Taken altogether, macrophage-STAT6 promotes cell migration and proliferation in lung cancer.

Drosophila TRP is a calcium-permeable cation channel essential for fly visual signal transduction. During phototransduction, Ca2+ mediates both positive and negative feedback regulation on TRP channel activity, possibly via binding to calmodulin (CaM). However, the molecular mechanism underlying Ca2+ modulated CaM/TRP interaction is poorly understood. Here, we discover an unexpected, Ca2+-dependent binding mode between CaM and TRP. The TRP tail contains two CaM binding sites (CBS1 and CBS2) separated by an ∼70-residue linker. CBS1 binds to the CaM N-lobe and CBS2 recognizes the CaM C-lobe. Structural studies reveal the lobe-specific binding of CaM to CBS1&2. Mutations introduced in both CBS1 and CBS2 eliminated CaM binding in full-length TRP, but surprisingly had no effect on the response to light under physiological conditions, suggesting alternative mechanisms governing Ca2+-mediated feedback on the channel activity. Finally, we discover that TRPC4, the closest mammalian paralog of Drosophila TRP, adopts a similar CaM binding mode.

Circular RNAs (circRNAs) are covalently closed single-stranded RNA molecules. After derived from precursor mRNA back-splicing, circRNAs play important roles in many biological processes. Recently, it was shown that several circRNAs were enriched in the mammalian brain with unclear functions. The expression of circRtn4 in the mouse brain was increased with the differentiation of primary neurons. In our study, knockdown of circRtn4 inhibited neurite growth, while overexpression of circRtn4 significantly increased neurite length. By dual-luciferase reporter assay and RNA antisense purification assay, circRtn4 was identified as a miRNA sponge for miR-24-3p. Moreover, knockdown of miR-24-3p increased neurite length, while overexpression of miR-24-3p significantly inhibited neurite growth. Furthermore, CHD5 was confirmed to be a downstream target gene of miR-24-3p. And CHD5 silence counteracted the positive effect of circRtn4 overexpression on neurite growth. In conclusion, circRtn4 may act as the sponge for miR-24-3p to promote neurite growth by regulating CHD5.

The study is aimed at investigating the changes in expressions of heat shock protein 27 (HSP27), HSP70, and soluble glycoprotein (SGP) in heart failure (HF) rats complicated with pulmonary edema and exploring their potential correlations with cardiopulmonary functions. The rat model of HF was established, and the rats were divided into HF model group (model group, n = 15 ) and normal group ( n = 15 ). After successful modeling, MRI and ECG were applied to detect the cardiac function indexes of the rats. The myocardial function indexes were determined, the injury of myocardial tissues was observed via hematoxylin and eosin (HE) staining, and the content of myeloperoxidase (MPO), matrix metalloproteinase-9 (MMP-9), and tumor necrosis factor-alpha (TNF-α) in the blood was measured. The partial pressure of oxygen (PaO2) and oxygenation index (OI) were observed, and the airway resistance and lung compliance were examined. Moreover, quantitative polymerase chain reaction (qPCR) and Western blotting assay were performed to detect the gene and protein expression levels of HSP27, HSP70, and SGP130. The levels of serum creatine kinase (CK), creatine (Cr), and blood urea nitrogen (BUN) were increased markedly in model group ( p < 0.05 ). Model group had notably decreased fractional shortening (FS) and ejection fraction (EF) compared with normal group ( p < 0.05 ), while the opposite results of left ventricular end-diastolic diameter (LVEDD) and left ventricular end-systolic diameter (LVESD) were detected. In model group, the content of serum MPO, MMP-9, and TNF-α was raised remarkably ( p < 0.05 ), OI and PaO2 were reduced notably ( p < 0.05 ), the airway resistance was increased ( p < 0.05 ), and the lung compliance was decreased ( p < 0.05 ). Obviously elevated gene and protein expression levels of HSP27, HSP70, and SGP130 were detected in model group ( p < 0.05 ). The expressions of HSP27, HSP70, and SGP130 are increased in HF rats complicated with pulmonary edema, seriously affecting the cardiopulmonary functions of the rats.

Blood-brain barrier (BBB) dysfunction is associated with an accumulation of neurotoxic molecules and increased infiltration of peripheral cells within the brain parenchyma. Accruing evidence suggests that microglia and astrocytes play a crucial role in the recovery of BBB integrity and the corralling of infiltrating cells into clusters after brain damage, but the mechanisms involved remain unclear. Intriguingly, the results of flow cytometry and immunofluorescence analyses have shown that BBB permeability to peripheral cells is substantially enhanced during normal aging at 12 months in mice. Thus, we used the SMART-seq2 method to perform RNA sequencing of microglia and astrocytes at five time points before and immediately after the BBB permeability change. Our comprehensive analyses revealed that microglia are characterized by marked alterations in the negative regulation of protein phosphorylation and phagocytic vesicles, whereas astrocytes show elevated enzyme or peptidase-inhibitor activity in the recovery of BBB function. Moreover, we identified a cassette of key genes that might ameliorate the insults of pathophysiological events in aging and neurodegenerative disease.

Objective: We aim to explore the expression of SOX9 in adenocarcinoma tissue at the esophagogastric junction and its correlation with clinicopathological features and prognosis.Methods: From January 2013 to May 2014, 114 samples of AEG tissues and 72 samples of normal paracancerous tissues at the surgical margin from the Department of Pathology at our hospital were collected. Using immunohistochemistry, we examined the esophagogastric junction adenocarcinoma (AEG) samples and compared their expression levels of human sex-determining region Y box protein 9 (SOX9) to SOX9 expression levels in adjacent cancer tissues. We further analyzed the correlation between SOX9 expression levels and clinicopathological characteristics and prognosis.Results: The high expression rate of SOX9 in AEG tissues was 46.49% (53/114), which was significantly higher than the low expression rate of SOX9 in adjacent tissues- 8.33% (6/72). The expression of SOX9 was correlated with the Lauren classification, tumor invasion depth, lymph node metastasis, distant metastasis, and the TNM stage in patients with AEG (P<0.05). It was not related to the patients' sex, age, tumor location, or tumor diameter tissue differentiation (P>0.05). According to Kaplan-Meier analysis, the 5-year survival rate of patients with high SOX expression was 30.19% (16/53), which was significantly lower than the 5-year survival rate of patients with low SOX expression (54.10%; 33/61). COX multivariate analysis revealed that the degree of tissue differentiation, depth of infiltration, and distant metastasis were independent risk factors affecting the prognosis of patients with AEG.Conclusion: SOX9 is highly expressed in AEG tissues. Its expression level is related to the occurrence, development, invasion, and metastasis of AE. While it affects the prognosis, SOX9 cannot be used as an independent risk factor for AEG patients.

目的 探讨CircCpsf6在N2a细胞中对细胞活力的影响,同时对CircCpsf6结合蛋白进行功能富集分析,预测CircCpsf6影响细胞活力的分子机制.方法 在N2a细胞中转染CircCpsf6的siRNA或过表达质粒,对Circ-Cpsf6进行敲减或过表达,用CCK-8法检测细胞活力.在CircCpsf6的接口处设计特异性探针,进行下拉实验,即CircCpsf6的反义纯化实验.经过磁珠吸附,蛋白的洗脱、纯化,蛋白的质谱检测,得到CircCpsf6可能结合的蛋白.利用在线网站STRING对这些蛋白之间可能存在的相互作用进行预测,同时利用在线网站DAVID进行蛋白功能富集分析.结果 在N2a细胞中,CircCpsf6能够被有效的敲减或过表达(P＜0.01),敲减CircCpsf6后,细胞活力上升(P＜0.01);过表达CircCpsf6后,细胞活力下降(P＜0.05).经过CircCpsf6的反义纯化实验,发现CircCpsf6可能与385个蛋白结合.GO分析和KEGG富集分析提示CircCpsf6可能通过结合蛋白影响细胞的增殖或者凋亡,从而影响细胞活力.结论 CircCpsf6能够抑制N2a细胞的细胞活力.CircCpsf6可能通过结合G6pdx、Pcx、Csl影响细胞增殖,通过结合2210010C04Rik、Try10、Gm2663影响细胞凋亡.

Objective. This study aims to investigate the effect of heat shock protein-70 (Hsp70) on epithelial-mesenchymal transition (EMT) of lung cancer cells under heat stimulation and to explore its possible molecular mechanism. Methods. qRT-PCR and immunohistochemistry assay were used to detect the expression of Hsp70 in lung cancer tissues and adjacent tissues. EdU assay was used to detect the cell activity. The effect of Hsp70 on the migration and invasion of A549 and NCI-H446 cells was detected by the wound-healing assay and Transwell assay. A tumor transplantation animal model was established to detect the effect of overexpression of Hsp70 on proliferation and metastasis of lung cancer cells. Western blot assay was used to detect the effect of thermal stimulation and overexpression of Hsp70 on SUMO modification of HIF-1α. Results. The wound-healing rate of A549 and NCI-H446 cells under Hsp70 stimulation was significantly higher than blank control group. At the same time, the number of cells passing through the membrane increased significantly. Hypodermic tumor transplantation in nude mice proved that knockout Hsp70 can inhibit proliferation and metastasis of lung cancer cells. Thermal stimulation upregulated the expression of Hsp70 and promoted SUMO modification of HIF-1α, ultimately promoting the proliferation and metastasis of lung cancer. Inhibition of Hsp70 reverses the effect of thermal stimulation on lung cancer by reducing the SUMO modification of HIF-1α. Conclusion. Thermal stimulation can promote EMT in A549 and NCI-H446 cells and promote cell migration and invasion in vitro and in vivo by upregulation of Hsp70. This process is associated with the promotion of SUMO modification of HIF-1α.

Radiofrequency ablation produces a heat-tolerance effect and increases HIF-1αp, and HSP70 expression is distributed in the lesion, but whether HSP70 mediates HIF-1α SUMOylation in lung cancer cells remains unclear. Mouse lung cancer LLC cells were cultured under hypoxia and randomly assigned into control group, heat tolerance group and HSP70 siRNA group followed by analysis of HSP70 and HIF-1α level by real time PCR and Western blot, association of HIF-1α with SUMO-1 and SUMO-2/3 by immunoprecipitation, SENP-1, Ubc9 and E3 ligase expression. CD4 + T cells were isolated and divided into control group, hyperbaric oxygen group, normal temperature hypoxia group, and high temperature hypoxia group followed by measurement of T17 and Treg cell by flow cytometry, and HIF-1α level. HSP70 and HIF-1α level was increased in heat tolerance group and reduced by HSP70 siRNA. Meanwhile, HSP70 siRNA decreased HSP70 binding to SENP-1, Ubc9, and E3 ligase. Heat tolerance group showed decreased SENP-1 expression, increased Ubc9 and E3 ligase expression. HIF-1 bound to SUMO-1, but not SUMO-2/3. HIF-1α expression was increased in CD4+ T cells in treatment group, with significantly increased CD4+ T cells apoptosis and changes of Treg and Th17 compared to control (P < 0 05). HSP70 can promote the heat tolerance effect of lung cancer cells by promoting SUMO-1 expression of HIF-1α; the heat tolerance effect leads to abnormal cellular immune response, which may affect the therapeutic effect.

目的 探讨EphB2在神经元神经突生长中的作用.方法 取EphB2单个等位基因敲除组KO WT组和两个等位基因敲除组KO KO组的C57BL/6胎鼠神经元体外分别培养1d和3d;采用实时荧光定量PCR法检测EphB2 mRNA相对表达量,免疫荧光法检测神经突生长情况,并与野生型基因组WT WT组比校.结果 KO WT和KO KO的EphB2 mRNA相对表达量(分别为0.66±0.12和0.36±0.03)均低于WT WT(1.00±0.14),差异有统计学意义(P＜0.05);且KO WT组和KO KO组比较差异有统计学意义(P＜0.05).神经元体外培养到第1天时,KO WT组和KO KO组细胞分支数(分别为4.630±1.462和4.314±1.855)均低于WT WT组(4.700±1.760),3组间差异无统计学意义(P＞0.05);KO WT组和KO KO组最长神经突长度[分别为(34.93±15.80)和(33.00±15.12) μm]均低于WT WT组[(42.96±20.16) μm],差异有统计学意义(P＜0.05);KO WT组和KO KO组神经突总长度[分别为(105.5±41.91) μm、92.24±47.87) μm]均低于WT WT组[(129.5±56.03) μm],差异有统计学意义(P＜0.05).神经元体外培养到第3天时KO WT组和KO KO组细胞分支数(分别为4.45±1.01和4.02±1.19)均低于WT WT组(5.42±1.07),差异有统计学意义(P＜0.05),且KO WT组和KO KO组比较差异也有统计学意义(P＜0.05);KO WT组和KO KO组最长神经突长度[分别为(230.2±125.7) μm和(150.3±87.84) μm]均低于WT WT组[(399.3±162.9) μm],差异有统计学意义(P＜0.05),且KO WT组和KO KO组比较差异也有统计学意义(P＜0.05);KO WT组和KO KO组神经突总长度[分别为(359.7±151.2)和(270.5±114.8) μm]均低于WT WT组[(586.8±178.8) μm],差异有统计学意义(P＜0.05),且KO WT组和KO KO组比较差异也有统计学意义(P＜0.05).结论 EphB2缺失后可能对神经突生长具有抑制作用.

Microglia and astrocytes, the two innate cells in CNS, are thought to protect and remodel of synapses for proper maintenance and plasticity of neuronal circuits. The two types of cells are the major responders by producing and releasing inflammatory mediators. Isolation of microglia and astrocytes from CNS tissue provides a powerful tool to study basic cell biology and examine the effects of in vivo treatments on microglia and astrocytes immunophenotype and function. The widely used approach of enrichment microglia and astrocytes from CNS was MACS (Magnetic activated cell sorting) and FACS (Fluorescence activated cell sorting). Here we described an optimized protocol of enzymatic dissociation generating single cell suspensions from brain tissue. Then the ability of the two methods to isolate microglia and astrocytes from brain dissociated cells was compared. Both MACS and FACS processing could obtain microglia and astrocytes with high viability (>85%). Microglia sorted by MACS comprises a slight myeloid cells contamination but with a little bit higher efficiency than that sorted by FACS. MACS processing was faster than FACS for either single or multiple samples. ACSA2 can be used to isolate astrocytes from both postnatal and adult brain, and is more suitable for purify astrocytes from newborn. FACS could get purer microglia which is helpful for deep sequencing and other related research. ACSA2 is a good marker of astrocytes.

The thymus plays an irreplaceable role as a primary lymphoid organ. However, the complicate processes of its development and involution are incompletely understood. Accumulating evidence indicates that non-coding RNAs play key roles in the regulation of biological development. At present, the studies of the circRNA profiles and of circRNA-associated competing endogenous RNAs (ceRNAs) in the thymus are still scarce. Here, deep-RNA sequencing was used to study the biological mechanisms underlying the development process (from 2-week-old to 6-week-old) and the recession process (from 6-week-old to 3-month-old) of the mouse thymus. It was found that 196 circRNAs, 233 miRNAs and 3807 mRNAs were significantly dysregulated. The circRNA-associated ceRNA networks were constructed in the mouse thymus, which were mainly involved in early embryonic development and the proliferation and division of T cells. Taken together, these results elucidated the regulatory roles of ceRNAs in the development and involution processes of the mouse thymus.