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Hypoxia-ischemia in the immature rodent brain impairs serotonergic neuronal function in certain dorsal raphé nuclei

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Neural Regeneration Research 2020年第3期15卷 457-463页

作者： Hanna E.Reinebrant Julie A.Wixey Kathryn M.Buller UQ Centre for Clinical Research Faculty of Medicine The University of Queensland

Neonatal hypoxia-ischemia(HI)results in losses of serotonergic neurons in specific dorsal raphe nuclei.However,not all serotonergic raphe neurons are lost and it is therefore important to assess the function of remaining neurons in order to understand their potential to contribute to neurological disorders in the HI-affected neonate.The main objective of this study was to determine how serotonergic neurons,remaining in the dorsal raphe nuclei after neonatal HI,respond to an external stimulus(restraint stress).On postnatal day 3(P3),male rat pups were randomly allocated to one of the following groups:(i)control+no restraint(n=5),(ii)control+restraint(n=6),(iii)P3 HI+no restraint(n=5)or(iv)P3 HI+restraint(n=7).In the two HI groups,rat pups underwent surgery to ligate the common carotid artery and were then exposed to 6%O2 for 30 minutes.Six weeks after P3 HI,on P45,rats were subjected to restraint stress for 30 minutes.Using dual immunolabeling for Fos protein,a marker for neuronal activity,and serotonin(5-hydroxytrypamine;5-HT),numbers of Fos-positive 5-HT neurons were determined in five dorsal raphe nuclei.We found that restraint stress alone increased numbers of Fos-positive 5-HT neurons in all five dorsal raphe nuclei compared to control animals.However,following P3 HI,the number of stress-induced Fos-positive 5-HT neurons was decreased significantly in the dorsal raphe ventrolateral,interfascicular and ventral nuclei compared with control animals exposed to restraint stress.In contrast,numbers of stress-induced Fos-positive 5-HT neurons in the dorsal raphe dorsal and caudal nuclei were not affected by P3 HI.These data indicate that not only are dorsal raphe serotonergic neurons lost after neonatal HI,but also remaining dorsal raphe serotonergic neurons have reduced differential functional viability in response to an external stimulus.Procedures were approved by the University of Queensland Animal Ethics Committee(UQCCR958/08/NHMRC)on February 27,2009.

关键词： dorsal raphe nuclei Fos hypoxia-ischemia neonate newborn brain injury preterm restraint stress serotonin

Transcriptomic analyses of humans and mice provide insights into depression

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Zoological Research 2020年第6期41卷 632-643页

作者： Hui-Juan Li Xi Su Lu-Wen Zhang Chu-Yi Zhang Lu Wang Wen-Qiang Li Yong-Feng Yang Lu-Xian Lv Ming Li Xiao Xiao Key Laboratory of Animal Models and Human Disease Mechanisms of the Chinese Academy of Sciences and Yunnan Province Kunming Institute of ZoologyChinese Academy of SciencesKunmingYunnan 650223China Kunming College of Life Science University of Chinese Academy of SciencesKunmingYunnan 650204China Henan Mental Hospital Second Affiliated Hospital of Xin xiang Medical UniversityXinxiangHenan 453002China Henan Key Lab of Biological Psychiatry International Joint Research Laboratory for Psychiatry and Neuroscience of HenanXinxiang Medical UniversityXinxiangHenan 453002China Henan Province People's Hospital ZhengzhouHenan 450003China KIZ-CUHK Joint Laboratory of Bioresources and Molecular Research in Common Diseases Kunming Institute of ZoologyChinese Academy of SciencesKunmingYunnan 650223China

Accumulating studies have been conducted to identify risk genes and relevant biological mechanisms underlying major depressive disorder(MDD).In particular,transcriptomic analyses in brain regions engaged in cognitive and emotional processes,e.g.,the dorsolateral prefrontal cortex(DLPFC),have provided essential insights.Based on three independent DLPFC RNA-seq datasets of 79 MDD patients and 75 healthy controls,we performed differential expression analyses using two alternative approaches for cross-validation.We also conducted transcriptomic analyses in mice undergoing chronic variable stress(CVS)and chronic social defeat stress(CSDS).We identified 12 differentially expressed genes(DEGs)through both analytical methods in MDD patients,the majority of which were also dysregulated in stressed mice.Notably,the mRNA level of the immediate early gene Fos(Fos proto-oncogene)was significantly decreased in both MDD patients and CVS-exposed mice,and CSDSsusceptible mice exhibited a greater reduction in Fos expression compared to resilient mice.These findings suggest the potential key roles of this gene in the pathogenesis of MDD related to stress exposure.Altered transcriptomes in the DLPFC of MDD patients might be,at least partially,the result of stress exposure,supporting that stress is a primary risk factor for MDD.

关键词： Major depressive disorder Stressed mice Dorsolateral prefrontal cortex Transcriptomic analysis Fos

Effects of plant-derived analgesic compounds sinomenine and salvinorin A in infant rats

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Journal of Integrative Medicine 2020年第2期18卷 174-180页

作者： Conrad J.Mascarenhas Renyu Liu Gordon A.Barr Department of Anesthesiology and Critical Care Children’s Hospital of PhiladelphiaPhiladelphiaPA 19104USA Department of Anesthesiology and Critical Care Perelman School of Medicine at the University of PennsylvaniaPhiladelphiaPA 19104USA Department of Psychology University of PennsylvaniaPhiladelphiaPA 19104USA

Objective: Premature and ill neonates undergo painful but medically necessary procedures while hospitalized. Although opiate drugs are administered as analgesics, problems associated with their side effects,tolerance, and potential dependence necessitate research into alternative pain-relieving medications.Here we test two plant-derived compounds in infant rats: sinomenine, which targets the Mas-related G-protein-coupled receptor member X2 opioid receptor;and salvinorin A, which is a j opioid receptor agonist. In adult animals both sinomenine and salvinorin A are analgesic, but neither has been tested in infants.Methods: We used the formalin and thermal plantar tests in rats 7 and 21 days of age(PN7 and PN21) for behavioral signs of pain. In addition, brain sections were stained using Fos immunohistochemistry to examine patterns of brain activation in the midbrain periaqueductal gray and the paraventricular nucleus of the hypothalamus.Results: Sinomenine was analgesic in both the formalin and thermal tests on animals 21 days of age. At PN7 only the highest dose elevated response latencies in the thermal test and there were no effects of sinomenine in the formalin test. Analysis of Fos expression in the sinomenine-treated animals showed no drug effect, in contrast to the behavioral results. Salvinorin A was analgesic in the formalin test only at the highest dose at 21 days of age but not in the thermal test at either age.Conclusion: The increased modest effectiveness of sinomenine in older animals and the minimum salvinorin A drug effect suggest that the compounds act on sites that develop during the preweaning period(sinomenine) or after weaning(salvinorin A).

关键词： Thermal pain Formalin test Fos Ontogeny Sinomenine Salvinorin A

后肢去负荷大鼠前扣带回皮质神经元Fos表达的时间模式

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神经解剖学杂志 2023年第1期39卷 1-7页

作者：张卓超金坤张勇张婷李辉张富兴董加强空军军医大学人体解剖与组织胚胎学教研室暨梁銶琚脑研究中心西安710032 解放军总医院第一医学中心肝胆胰外科医学部北京100000 空军军医大学基础医学院外语教研室西安710032 空军军医大学第一附属医院消化内科西安

目的:研究模拟失重模型动物前扣带回皮质(anterior cingulated cortex,ACC)神经元激活状况及其变化的时间模式。方法:将SD大鼠随机分成4组。所有动物采用吊尾、头低位方式使后肢去负荷(hind-limb unloading,HU)方式模拟失重生理。第一... 详细信息

目的:研究模拟失重模型动物前扣带回皮质(anterior cingulated cortex,ACC)神经元激活状况及其变化的时间模式。方法:将SD大鼠随机分成4组。所有动物采用吊尾、头低位方式使后肢去负荷(hind-limb unloading,HU)方式模拟失重生理。第一、二组分别经HU处理1 d和4 d。第三、四组动物分别经过HU 1 d和4 d,并进行双轴旋转运动刺激2 h。然后,动物灌流固定、取脑、切片(厚度25μm),采用常规免疫组织化学染色技术卵白素-生物素复合物(ABC)法对ACC内神经元Fos蛋白进行免疫反应,3,3′-二氨基联苯胺四盐酸盐水合物(DAB)为呈色剂。统计分析Fos阳性神经元数量变化。结果:(1)1-d HU组、4-d HU和1-d HU-旋转组和4-d HU-旋转组动物ACC内Fos阳性神经元数目分别是344.98±90.620、389.111±133.774、486.47±182.152和464.444±286.881。(2)HU处理1 d到4 d ACC神经激活数目有增加趋势,旋转刺激后ACC内神经元也有被激活趋势,但统计分析显示各组间无统计学差异。结论:HU处理使ACC内神经元有被激活趋势,但随HU处理时间的延长,ACC神经元活动对HU刺激产生适应。

关键词： Fos 后肢去负荷扣带前回皮质免疫组织化学大鼠

骨母细胞瘤临床病理分析以及Fos基因融合在肿瘤中的作用机制研究

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骨母细胞瘤临床病理分析以及FOS基因融合在肿瘤中的作用机制研究

作者：耿亚飞中国人民解放军海军军医大学

学位级别：硕士

研究背景骨母细胞瘤(Osteoblastoma)是一种交界性成骨细胞肿瘤,多见于10-25岁人群。脊柱背侧附件是骨母细胞瘤的好发部位,占所有病例的32%到45%。骨母细胞瘤具有局部侵袭性生长方式以及潜在骨质破坏作用,必须要行外科手术切除。该病具... 详细信息

研究背景骨母细胞瘤(Osteoblastoma)是一种交界性成骨细胞肿瘤,多见于10-25岁人群。脊柱背侧附件是骨母细胞瘤的好发部位,占所有病例的32%到45%。骨母细胞瘤具有局部侵袭性生长方式以及潜在骨质破坏作用,必须要行外科手术切除。该病具有一定的复发倾向,复发率为24%。个别病例在复发时可恶性进展为骨肉瘤,预后很差。近年来多项研究证实Fos基因融合突变在骨母细胞瘤中广泛存在,但Fos融合基因与该病的发生、进展和复发的关系以及作用机制尚不明确。第一部分:骨母细胞瘤临床与病理学资料分析研究方法收集分析临床2012—2020年骨母细胞瘤患者病历,影像学资料、实验室检验结果;HE及IHC染色明确诊断;并对患者进行随访,Kaplan-Meier法、COX风险回归模型评估影响患者预后PFS的因素。研究结果1.骨母细胞瘤好发于11-30岁(60.7%),发病平均年龄为26.1岁;男女比例为1.2:1;颈椎占所有病例的36%,胸椎占35%,腰骶椎占21%,脊柱外病例仅占8%;肿瘤平均大小为3.47cm(0.7cm到9.5cm);2.上皮样骨母细胞瘤的肿瘤体积更大(P=0.001),继发ABC改变更常见(P=0.019);发病年龄>30岁组的复发率显著高于11-30岁组(P=0.014);Curettage手术组患者的复发率显著高于En-bloc手术组(P=0.035);3.上皮样骨母细胞瘤分型(P=0.015,HR=4.298,95%CI:1.326-13.933)、发病年龄大于30岁(P=0.017,HR=4.346,95%CI:1.305-14.470)是患者PFS的独立危险因素;手术方式不是预后的独立影响因素(P=0.065)。结论骨母细胞瘤患者的发病年龄≤30岁、En-bloc手术组的复发率显著降低;发病年龄大于30岁、上皮样骨母细胞瘤的病理分型是骨母细胞瘤术后无进展生存期的独立危险因素。第二部分:骨母细胞瘤样本的RNA测序与验证研究方法收集20例骨母细胞瘤患者的石蜡包埋肿瘤样本,经病理学鉴定后提取总核酸进行RNAseq;以GRCh38为对照基因组,筛选可能的样本融合基因;在未脱钙石蜡组织切片上行免疫组化Fos蛋白染色、FISH对Fos基因融合的情况进行验证;并对样本JDP2蛋白表达情况行IHC检测。研究结果1.在20例样本中,共5例样本提示基因融合,融合发生率为25%;Fos-JDP2是最常见的基因融合形式;上皮样骨母细胞瘤和经典型骨母细胞瘤中的Fos基因融合不存在显著差异(P=0.175);在肿瘤≥4cm和Fos-JDP2基因融合突变的肿瘤组织中JDP2的蛋白表达显著下降(PFos蛋白核表达增强;无论是否存在基因融合FISH检测结果均呈阴性。结论1.Fos-JDP2基因融合发生率为25%,与患者的病理分型、肿瘤大小无关;2.Fos-JDP2融合突变的样本中JDP2的蛋白表达水平显著降低,Fos蛋白表达显著升高;FISH对Fos-JDP2基因融合与否无提示性。第三部分:细胞实验与蛋白质组学验证Fos-JDP2融合基因的作用研究方法在小鼠MC3T3-E1 Subclone 14细胞慢病毒转染Fos-JDP2融合序列构建稳定表达细胞株;对细胞的增殖曲线、细胞周期,对Fos基因的m RNA和蛋白表达水平进行测定;蛋白质组学测定组间差异表达蛋白,并对差异蛋白进行GO富集、KEGG富集、PPI和转录因子家族等分析;WB法对预测的目标蛋白进行验证。研究结果1.在MC3T3-E1 Subclone 14细胞中转染Fos-JDP2融合基因可以使细胞增殖加快,细胞周期S期的细胞比例增多,Fos基因的m RNA和蛋白的表达水平显著升高;野生型Fos基因可以和融合突变Fos基因可以产生相似的效应,但Fos蛋白水平变化没有突变组显著;2.HMGA2蛋白在Fos-JDP2融合突变细胞与Fos过表达细胞中表达均显著升高(PFos-JDP2融合突变可以促进成骨细胞的增殖、细胞周期进入;促进Fos基因的转录和翻译水平;2.HMGA2可能是Fos-JDP2融合基因影响的下游关键蛋白。

关键词：骨母细胞瘤 Fos 基因融合 JDP2 HMGA2

创伤后应激障碍小鼠海马恐惧记忆消退相关神经元的异常激活

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神经解剖学杂志 2023年第6期39卷 617-623页

作者：刘珺琛解进祎刘明月肖昊翔郭保霖武胜昔陕西中医药大学基础医学院咸阳712000 空军军医大学基础医学院神经生物学教研室西安710032 延安大学医学院生理学教研室延安716099

目的:分析创伤后应激障碍(PTSD)小鼠恐惧记忆消退过程中海马各亚区神经元的激活及差异。方法:使用两种即刻早期基因蛋白标记策略,对PTSD小鼠条件性恐惧记忆消退相关神经元进行标记。第一组利用免疫荧光染色技术,使用野生型小鼠对海马区... 详细信息

目的:分析创伤后应激障碍(PTSD)小鼠恐惧记忆消退过程中海马各亚区神经元的激活及差异。方法:使用两种即刻早期基因蛋白标记策略,对PTSD小鼠条件性恐惧记忆消退相关神经元进行标记。第一组利用免疫荧光染色技术,使用野生型小鼠对海马区域神经元Arc蛋白进行标记并观察;第二组使用Fos-CreER~(T2);Ai9转基因小鼠,在恐惧记忆消退前23 h注射他莫昔芬诱导荧光蛋白表达,标记相关神经元并观察。通过对标记的海马神经元数量及树突分支结构进行分析,对比海马神经元激活水平和神经元树突的可塑性变化。结果:两组实验观察发现Arc和Fos阳性神经元主要分布在背侧海马,其中Arc蛋白显色在CA3和DG亚区呈富集分布,CA1、CA2亚区的分布则较为散在,而Fos阳性神经元在海马DG亚区表达富集,散在分布于CA1、CA2和CA3亚区。与对照组对比,PTSD组海马各亚区表达Arc蛋白神经元数目没有明显差异;而PTSD组海马中CA1、CA3、DG亚区的Fos阳性神经元数量显著增加(PFos-CreER~(T2);Ai9小鼠对两组小鼠海马区域神经元树突分支进行观察分析,未发现明显变化。结论:PTSD小鼠恐惧记忆消退过程中海马不同亚区神经元存在异常激活,但激活的神经元树突分支无明显可塑性改变。

关键词：恐惧记忆消退创伤后应激障碍海马 Fos 小鼠

miR-150-5p靶向Fos调控EV-D68复制的研究

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miR-150-5p靶向Fos调控EV-D68复制的研究

作者：司俊卓重庆医科大学

学位级别：硕士

目的:利用EV-D68感染宿主细胞（实验组）并设置相应对照组,对两组进行全转录组高通量测序,随后进行生物信息学相关预测,挖掘miRNA和Fos的靶向关系;采用miRNA模拟体（mimic）与miRNA抑制体（inhibitor）转染宿主细胞验证miRNA对EV-D68是... 详细信息

目的:利用EV-D68感染宿主细胞（实验组）并设置相应对照组,对两组进行全转录组高通量测序,随后进行生物信息学相关预测,挖掘miRNA和Fos的靶向关系;采用miRNA模拟体（mimic）与miRNA抑制体（inhibitor）转染宿主细胞验证miRNA对EV-D68是否具有调控作用,并与Fos相结合探讨miR-150-5p/Fos这一轴对EVD68调控作用的机制。方法:（1）EV-D68感染人横纹肌瘤（Rhabdomyosarcoma,RD）细胞（实验组）并设置相应对照组,分别收集两组细胞,提取总RNA。随后对两组进行全转录组高通量测序分析,得到所对应的全转录组差异表达谱。筛选出差异表达基因,利用RT-q PCR技术验证测序结果。通过构建蛋白互作网络（protein-protein interaction,PPI）筛选关键m RNA（Hub m RNA）分子。通过在线靶基因数据库得到3种非编码RNA（miRNA、lnc RNA和circ RNA）与m RNA之间的相互靶向关系,构建出一个miRNA居于中心位置,其余非编码RNA以及m RNA处于周边的竞争性内源RNA（ce RNA）调控网络。并通过对此网络进一步的分析,挖掘出了与Fos相互靶向的miRNA。利用RT-q PCR验证此miRNA在EV-D68感染后其表达水平的变化。（2）通过miR-150-5p mimic（inhibitor）转染RD细胞,改变miRNA表达水平,RT-q PCR技术观察Fos表达水平是否出现相应变化,随后利用病毒滴度TCID50测定,RT-q PCR、Western blot等技术,观察EV-D68复制水平的变化。利用双荧光素酶报告实验确定miR-150-5p与Fos之间的靶向关系。用miR-150-5p mimic（inhibitor）与CD513B-Fos（si-Fos）共转染细胞,RT-q PCR和Western blot技术检测EV-D68 VP1 RNA和蛋白表达水平,进一步确定miR-150-5p表达变化对Fos调控EV-D68复制的影响。结果:（1）EV-D68感染RD细胞经全转录组高通量测序分析得到239个差异表达m RNA（DEm RNAs）,96个差异表达miRNA（DEmiRNAs）,375个差异表达lnc RNA（DElnc RNAs）以及33个差异表达circ RNA（DEcirc RNAs）,并通过RT-q PCR验证了4个DEm RNAs以及6个DEmiRNAs,其表达趋势与测序结果基本一致。利用GO与KEGG富集分析发现:GO富集分析结果主要涉及RNA聚合酶II转录调控、蛋白质信号转导以及DNA结合转录因子活性等功能。KEGG信号通路富集分析主要富集在MAPK、Hippo、AGERAGE以及Apoptosis信号通路上。通过构建PPI互作网络筛选出以EGR1、Fos以及Jun为主的Hub m RNA分子。通过靶向关系的预测,构建出以miRNA为核心的ce RNA调控网络,并从中筛选出了与Fos靶向的miRNA分子即miR-150-5p。RT-q PCR结果显示EV-D68感染RD细胞后其表达水平下调,且与病毒感染时间及感染复数呈正相关。（2）利用miR-150-5p mimic（inhibitor）分别转染RD细胞后,miR-150-5p表达水平显著上升（下降）,与其相对应Fos的表达水平显著下降（上升）,与此同时,EV-D68在RD细胞中的复制明显得到促进（抑制）。通过双荧光素酶报告实验,成功的验证了miR-150-5p靶向Fos这一关系的存在。通过共转染miR-150-5p mimic（inhibitor）与CD513B-Fos（si-Fos）至RD细胞内,发现miR-150-5p能够通过表达量的改变影响Fos的表达水平从而间接调控EV-D68的复制。结论:本研究通过高通量测序分析结果构建了ce RNA调控网络,并挖掘出miR-150-5p与Fos之间靶向关系的存在。研究结果表明,miR-150-5p通过靶向Fos表达,调控EV-D68在宿主细胞的复制。

关键词：肠道病毒D68型全转录组测序 miR-150-5p Fos 病毒复制

Insight into the structural features of organic species in Fushun oil shale via thermal dissolution

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Chinese Journal of Chemical Engineering 2018年第10期26卷 2162-2168页

作者： Shengkang Wang Xianyong Wei Zhimin Zong Key Laboratory of Coal Processing and Efficient Utilization Ministry of Education China University of Mining & Technology State Key Laboratory of High-efficiency Coal Utilization and Green Chemical Engineering Ningxia University

Fushun oil shale(Fos) was subjected to thermal dissolution(TD) under different conditions. The results show that the optimal solvent, temperature, time, and ratio of solvent to Fos are ethanol, 300 °C, 2 h, and 5 ml·g^(-1),respectively and the corresponding yield of the soluble portion(SP) is 32.2%(daf), which is much higher than the oil content of Fos(ca. 6%), suggesting that TD in ethanol is an excellent way to extract organics from Fos.According to 3 direct analyses, aliphatic moieties in Fos are the most abundant followed by C\\O-containing moieties and each cluster in Fos has 3 conjugated aromatic rings on average with fewer substituents. According to the analysis with a gas chromatograph/mass spectrometer, alkanes are predominant in all the SPs. A number of alkenes were identified in the SPs from the TD, while none of the alkenes were detected in acetone-SP obtained at room temperature, implying that the TD can destroy the π-π and intertwining interactions between alkenes and macromolecular structures in Fos. Moreover, a small amount of alkyl-substituted phenols and alkoxysubstituted phenols were detected in ethanol-SP from the TD, which could be the products from ethanolyzing the macromolecular moiety of Fos.

关键词：热溶解油页岩结构特征种类器官房间温度 Fos

三阴性乳腺癌关键基因Fos和SPP1的生物信息学分析

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中国免疫学杂志 2021年第12期37卷 1454-1460,1467页

作者：谭玉靓唐标湖南中医药大学医学院长沙410208

目的:基于生物信息学理论,从基因和通路层面分析三阴性乳腺癌(TNBC)的发病机制,揭示TNBC的关键致病基因,为TNBC的诊治和评估提供新思路。方法:通过GEO数据库下载TNBC细胞系的基因芯片信息和微矩阵数据,基于P值及|log2FC|值对GEO原始数... 详细信息

目的:基于生物信息学理论,从基因和通路层面分析三阴性乳腺癌(TNBC)的发病机制,揭示TNBC的关键致病基因,为TNBC的诊治和评估提供新思路。方法:通过GEO数据库下载TNBC细胞系的基因芯片信息和微矩阵数据,基于P值及|log2FC|值对GEO原始数据进行筛选,确定TNBC相关差异表达基因(DEGs)。利用David和KOBAS在线分析平台分别对获得的DEGs进行富集分析;利用STRING在线平台构建DEGs的蛋白质相互作用(PPI)网络。利用cytoscape的cytoHubba软件,得到关键基因。利用GEPIA在线平台,分析关键基因在TNBC不同病理阶段的异常表达。利用Kaplan-Meier Plotter在线分析工具,分析关键基因对TNBC患者预后的影响。结果:本研究共筛选得到61个DEGs。GO富集分析显示,这些DEGs参与正向调控血管内皮生长因子(VEGFs)、细胞迁移、G1/S期转换等生物过程。KEGG富集分析显示,DEGs介导PI3K/Akt、P53以及炎症相关因子等多条信号通路。PPI网络显示DEGs之间具有联系;PPI网络的关键节点Fos和SPP1与患者预后相关。结论:DEGs介导的通路可能是TNBC的发病机制,关键基因Fos和SPP1与TNBC患者预后密切相关,或许可作为TNBC预后评估的生物标记。

关键词：三阴性乳腺癌差异表达基因 Fos SPP1