人鼻咽与鼻咽癌及肺癌基因表达谱差异的研究

研究鼻咽癌、肺癌与正常鼻咽组织基因表达谱差异及筛选鼻咽癌相关基因,采用α- ³²P逆转录标记组织总RNA,将cDNA探针与有5 184个基因或表达序列标签EST(expression sequence tag)的高密度cDNA微阵列GF200杂交,软件分析表达谱差异.结果发现三者均呈低表达为主的表达谱,密度值在200以上的基因及EST在鼻咽癌有110个,肺癌134个而鼻咽组织有158个;5个EST在鼻咽高表达但鼻咽癌低表达,3个EST在鼻咽癌高表达但正常鼻咽低表达.结果表明鼻咽癌与正常鼻咽及肺癌组织存在差异表达基因,可能还有新基因在鼻咽癌发生中起作用;采用高密度cDNA微阵列是一种筛选差异表达基因的快速有效方法.     

遗传性癫痫易感大鼠脑内NMDAR1基因表达

N-甲基-D天门冬氨酸(NMDA)受体与癫痫及癫痫易感性的形成密切相关. 以遗传性癫痫易感大鼠P77PMC为研究对象, 通过RNA印迹杂交检测,NMDA受体一型亚单位(NMDAR1)mRNA在惊厥后不同脑区表达, 结果显示: P77PMC大鼠惊厥后, 大脑皮层、海马、皮层下、下丘NMDAR1 mRNA表达呈时间依赖性增加;比较惊厥即刻与惊厥后24 h, 四个脑区NMDAR1 mRNA分别增加了111%、113%、165%和202%. 提示NMDA受体 亚单位受惊厥活动调控,并参与惊厥的发生、发展及惊厥后突触结构的重建.     

HSV转基因表达IL-1ra抑制P77PMC大鼠的惊厥发作

利用PCR基因克隆及基因转染技术 ,构建含人IL 1ra(IL 1receptorantago nist,IL 1ra)cDNA的质粒型单纯疱疹病毒 (herpessimplexvirus ,HSV)载体 ,再由HSV 1tsK株包装成假病毒颗粒 ,然后注入P77PMC听源性惊厥易感大鼠侧脑室 ,观察LacZ基因和IL 1ra在脑内的表达情况及对P77PMC大鼠惊厥程度的影响 .结果表明 :( 1 )LacZ在脑内神经和非神经细胞可持续表达 8周以上 ;( 2 )侧脑室注射pHSV IL 1ra假病毒可明显抑制P77PMC大鼠的惊厥发作 .原位杂交和免疫荧光染色发现脑膜和脉络丛上皮细胞及某些神经细胞有IL 1ramRNA和蛋白的表达 .结果表明HSV载体可有效地将IL 1ra基因转入脑内 ,并能明显抑制P77PMC听源性惊厥易感大鼠的惊厥发作 .     

青霉素致痫大鼠海马神经元单位放电特征分析

目的:观察青霉素致痫大鼠海马神经元单位放电特征。方法:24只Wistar雄性健康大鼠,随机分为正常对照组、癫痫模型组各12只。癫痫模型组用青霉素钠按6×106U/kg腹腔注射,观察癫痫发作级别,记录海马神经元单位放电。结果:正常对照组大鼠共记录到24个单位海马神经元放电,放电频率以中低频放电为主,放电形式以单个不均匀放电为主;癫痫模型组共记录到78个单位海马神经元放电,放电频率以高频放电为主,放电形式则以混合型放电为主,癫痫发作程度强。癫痫模型组与正常对照组比较,海马神经元单位放电数、放电频率以及放电形式均有显著性差异(P0.01);结论:青霉素诱导的癫痫模型,海马神经元单位放电数明显增多,放电频率高,并以混合型爆发放电为主。     

AGRIN在老年鼠大脑皮质和海马的表达

目的观察蛋白聚糖-agrin在大鼠大脑皮质和海马的表达,探讨agrin与记忆的关系.方法用抗agrin的多克隆抗体,免疫组化方法检测成年大鼠、记忆正常老年鼠以及记忆障碍的老年鼠大脑皮质和海马中agrin的表达变化.结果 Agrin在不同动物组的表达变化明显,在成年鼠和记忆正常的老年鼠的大脑皮质和海马agrin有较弱表达,但在记忆障碍的老年鼠其表达增加明显.结论 Agrin表达可能和大鼠的记忆障碍有关.     

戊四氮致痫大鼠大脑皮质和海马内GFAP和cyclin D1表达的变化及脑和脑脊液天冬氨酸和谷氨酸含量的变化

目的研究癫痫发病时星形胶质细胞的激活及脑和脑脊液兴奋性氨基酸含量的变化.方法将实验SD大鼠随机分为2组:1.生理盐水对照(control)组(n=8);腹腔注射与致痫剂等容量的生理盐水.2.戊四氮(PTZ)组;PTZ 60mg/kg腹腔注射后2h、4h、8h、12h取脑,每个时间点各8只,检测下列指标:(1)用免疫组织化学方法(SABC法)观察大鼠大脑皮质和海马内星形胶质细胞胶质原纤维酸性蛋白(GFAP)含量的变化;(2)用Western blot方法检测大鼠大脑皮质和海马细胞周期素D1(cyclin D1)表达的变化;(3)采用高效液相色谱(HPLC)法测定大鼠大脑皮质和海马组织匀浆及脑脊液中兴奋性氨基酸(天冬氨酸和谷氨酸)含量的变化.结果星形胶质细胞GFAP免疫组织化学染色结果显示癫痫发作4h后在大脑皮质和海马CA3区、CA1区和皮质内GFAP免疫反应明显增强(P<0.05);Western blot检测癫痫发作2h后cyclin D1的表达在皮质和海马均较对照组明显增强(P<0.05);HPLC测定癫痫发作4h后大脑皮质中天冬氨酸(Asp)和谷氨酸(Glu)的含量达到最高峰,分别为8.900±0.540μmol/g protein和17.500±0.526μmol/g protein,与对照组(分别为7.083±0.693μmol/g protein和7.017±0.419μmol/g protein)相比均有显著性差异(P<0.05),在海马内Asp的含量在癫痫发作后2h达到最高峰,为11.425±1.006μmol/g protein,Glu的含量在癫痫发作后4h达到最高峰,为17.698±0.250μmol/g protein,与对照组(分别为7.300±0.824μmol/g protein和10.925±0.323μmol/g protein)比较均有显著性差异(P<0.05),脑脊液中Asp的含量在癫痫发作后2h达到最高峰,为82.65±4.81μmol/L,而Glu的含量在癫痫发作后持续增高,12h后达到72.80±3.66μmol/L.结论戊四氮致痫过程中兴奋性氨基酸含量增多,海马及皮质内星形胶质细胞激活,cyclin D1表达增加,星形胶质细胞增殖,以上变化在癫痫的形成和维持中发挥作用.     

马桑内酯激活的星形胶质细胞条件培养液对正常大鼠脑内孕激素及其受体的影响

目的探讨星形胶质细胞对大鼠脑内孕激素及其受体的影响以及在癫痫发病中的作用。方法将马桑内酯(Coriaria lactone,CL)激活的星形胶质细胞条件培养液(Astrocytic conditioned medium,ACM)注射入正常SD大鼠侧脑室后,观察大鼠的行为变化;运用免疫组织化学方法观察大脑皮质及海马中孕激素受体(PR)表达的变化;运用放射免疫分析方法,观察脑组织匀浆及脑脊液内孕酮含量的变化。结果ACM组大鼠在注射ACM后30min出现癫痫行为,2h恢复正常;免疫组织化学显示:ACM作用后2h,PR免疫反应阳性神经元数和平均光密度值明显降低,4h达最低(P<0.05),12h恢复正常水平;放射免疫分析方法显示ACM组大鼠在侧脑室注射ACM后2h,脑脊液中孕酮含量明显升高;而海马组织和大脑皮质中孕酮含量则在注药后4h明显降低,与对照组比较均有显著差异(P<0.05)。结论以上实验结果提示马桑内酯激活的星形胶质细胞条件培养液可通过降低大鼠脑内孕激素及其受体的表达参与癫痫的反复发作。     

根据基因功能表达谱研究抑郁症模型及安佳欣胶囊的抗抑郁症机制

由基因芯片检测正常大鼠、模型大鼠和服用安佳欣胶囊进行治疗后的大鼠各8例的基因表达谱,在正常组和模型组之间,筛选得到207个差异表达基因和25个差异表达基因功能模块,在模型组和给药组之间,筛选得到860个差异表达基因和24个差异表达基因功能模块。比较两次结果,得到信号传导通路、蛋白质转运等价相同的功能模块,预测此功能模块可能为安佳欣胶囊发挥抗抑郁作用的一部分靶点。在这9个模块中寻找在模型组出现差异表达而在给药组表达趋于正常水平的基因,从基因水平分析药物的治疗机制。     

cDNA微阵列技术分析NAG7基因对鼻咽癌细胞基因表达谱的影响

运用cDNA微阵列技术分析NAG7基因重表达对HNE1细胞基因表达谱的影响.抽提HNE1细胞和pcDNA3.1(+)/NAG7/HNE1细胞总RNA,分离polyA mRNA,将mRNA逆转录为cDNA,并在逆转录过程中用³³P-dATP进行标记,与含有16 150个基因和表达序列标签(EST)的cDNA表达阵列膜杂交,获得基因表达图谱.Array Gauge软件分析NAG7基因的重表达所导致的鼻咽癌细胞HNE1基因表达谱改变,并用RNA印迹对微阵列杂交结果进行验证.结果分析表明,2倍以上的差异表达基因或EST 179个,其中表达上调的91个,表达下调的88个;已明确基因表达产物的上调基因29个,下调基因37个.在差异表达基因中,涉及基因转录调控、信号转导、细胞生长、细胞代谢和细胞凋亡等基因.RNA印迹证实生长阻滞特异蛋白1（gas 1）基因表达上调.特别值得关注的是, 先前的蛋白质组研究结果亦发现NAG7基因可导致生长阻滞特异蛋白1表达上调,说明gas 1基因在NAG7重表达的HNE1细胞中具有重要作用,这为深入研究NAG7基因的作用环节和机理提供了重要的线索.     

戊四氮致痫大鼠皮质和海马内GFAP、cyclinD1及脑和脑脊液17β-雌二醇和孕酮浓度变化

目的研究癫痫发作后脑和脑脊液中17β-雌二醇和孕酮含量的变化.方法将雌性SD大鼠随机分为戊四氮(PTZ)致痫组和生理盐水对照组.(1)用免疫组织化学方法观察大鼠大脑皮质和海马星形胶质细胞胶质原纤维酸性蛋白(GFAP)含量的变化;(2)用Western blot方法检测大鼠大脑皮质和海马细胞周期素D1(cyclin D1)表达的变化;(3)采用放免法测定大鼠大脑皮质和海马组织匀浆及脑脊液中17β-雌二醇和孕酮含量的变化.结果免疫组织化学染色结果显示癫痫发作4h后在海马CA3区、CA1区和皮质内GFAP免疫反应明显增强(P<0.05);Western blot结果显示癫痫发作2h后cyclin D1的表达在皮质和海马均较对照组明显增强(P<0.05);放射免疫分析结果显示,癫痫发作后,皮质、海马和脑脊液的17β-雌二醇的浓度有不同程度增高,致痫8h后恢复正常,孕酮的浓度在皮质和脑脊液则呈下降趋势.结论戊四氮致痫时皮质及海马内星形胶质细胞激活、增殖,内源性雌激素合成增多,同时孕酮浓度降低,说明雌性激素在癫痫的形成和维持中发挥作用.     

听源性遗传癫痫易感大鼠大脑皮层胆囊收缩素mRNA的原位杂交检测

本实验用原位杂交法,对听源性遗传癫痫易感大鼠（P77PMC)癫痫发作前,单次癫痫发作和多次发作时大脑颞皮层CCK mRNA阳性信号进行了检测,结果显示：（1）单次和多次癫痫发作后颞皮层CCK mRNA阳性神经元数量明显增加（P＜0．01）;（2）多次癫痫发作者上述脑区CCK mRNA阳性神经元数量较单次癫痫发作有明显的下降（P＜0．01）,大脑颞皮层CCK mRNA增高表明,CCK mRNA在癫痫发作过程中起了某种作用,多次癫痫发作大鼠CCK mRNA表达降低提示,单次和多次癫痫发作时大脑皮层CCK mRNAl转录的调控可能存在不同的机制。     

缺氧对大鼠大脑皮质细胞色素氧化酶亚基Ⅰ、Ⅳ表达协同性的影响

本文探讨缺氧对细胞色素氧化酶（cytochrome oxidase,COX,即complexⅣ）的mtDNA和nDNA编码亚基Ⅰ,Ⅳ表达及其协同性的影响。实验用成年雄性Wistar大鼠随机分为对照组,缺氧2,5,15和30d组,缺氧大鼠于低压舱内模拟海拔5000m连续减压,对照组大鼠于舱外同时喂养（舱外海拔高度为300m)。用半定量逆转录-PCR法测定大脑皮质COXⅠ,ⅣmRNA量,用Western blot分析大脑皮质线粒体COXⅠ,Ⅳ蛋白量,以两个亚基的蛋白量,mRNA量的比值反映亚基表达的协同性,结果显示,缺氧2,5d,COXⅠmRNA增加,缺氧15,30d时下降至对照水平,缺氧2,5和15d时,COXⅣmRNA显著增加,缺氧30d时降低,与对照组差异非常显著。COXⅣ,ⅠmRNA比值在缺氧15d时最高,其它各缺氧组与对照组的差异无显著意义。各组COXⅠ,Ⅳ蛋白量及其比值均无显著差异。上述结果表明,缺氧可影响COXⅠ,ⅣmRNA的表达及其协同性,但对蛋白的表达及其协同性没有显著影响,提示转录后调节是缺氧过程中线粒体内COXⅠ,Ⅳ蛋白表达协同的主要机制。     

Gene Expression Profiling of Rat Cerebral Cortex Development Using cDNA Microarrays

A large amount of genetic information is devoted to brain development. In this study, the cortical development in rats at eight developmental time points (four embryonic [E15, E16, E18, E20] and four postnatal [P0, P7, P14, P21]) was studied using a rat brain 10K cDNA microarray. Significant differential expression was observed in 467 of the 9,805 genes represented on the microarray. Two major Gene Ontology classes—cell differentiation and cell–cell signaling—were found to be important for cortical development. Genes for ribosomal proteins, heterogeneous nuclear ribonucleoproteins, and tubulin proteins were up-regulated in the embryonic stage, coincidently with extensive proliferation of neural precursor cells as the major component of the cerebral cortex. Genes related to neurogenesis, including neurite regeneration, neuron development, and synaptic transmission, were more active in adulthood, when the cerebral cortex reached maturity. The many developmentally modulated genes identified by this approach will facilitate further studies of cortical functions. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Role of Quercetin on PCBs (Aroclor-1254) Induced Impairment of Dopaminergic Receptor mRNA Expression in Cerebral Cortex of Adult Male Rats

The present study is aimed to evaluate the putative neuroprotective effect of quercetin on PCB induced impairment of dopaminergic receptor mRNA expression in cerebral cortex of adult male Wistar rats. Group I (control) received only vehicle (corn oil; 0.1 ml/kg bwt) intraperitoneally (i.p); Group II Aroclor 1254 at a dose of 2 mg/kg bwt/day (i.p); Group III (Aroclor 1254—exposed (i.p), quercetin treated gavage (50 mg/kg bwt/day); Group IV received quercetin alone (gavage). 24 h after the 30th day treatment rats were euthanized. From each rat cerebral cortex tissues was collected and analyzed for mean activities of creatine kinase, acetylcholine esterase, Na⁺/K⁺, Ca²⁺ and Mg²⁺ATPases, Hydrogen peroxide generation, protein carbonyl content and lipid peroxidation levels. The fates of the mRNA expression of dopaminergic receptors, Cacna1d on all the groups were studied by RT–PCR. Results evidenced that significant reduction of neurodegeneration in PCBs exposed rats treated with quercetin was ascertained suggesting, quercetin treatment precludes against PCB induced oxidative stress and protects dopaminergic receptor dysfunction in rat cerebral cortex.     

Effect of dDAVP, agonist of V2 vasopressin receptor on the hyaluronidase-1 and hyaluronidase-2 gene expression in the kidneys of Wistar and Brattleboro rats

Effect of vasopressin on the expression of Hyal-1 and Hyal-2 genes in different functional zones of Wistar and homozygous vasopressin-defficient Brattlboro rat kidneys was analysed using RT-PCR mehod. It was found that, in Wistar rats the content of Hyal-1 mRNA was higher in the medulla than in other kidney zones at the normal water and food regimen. The level of Hyal-1 mRNA in the cortex and the medulla of Brattlboro rat kidney exceeded that of papilla. There were no significant differences in the Hyal-2 mRNA content detected between functional zones of Wistar and Brattlboro rat kidneys. The treatment by dDAVP, the agonist of V2 vasopressin receptor (Desmopressin, 10 microg/100 g b.w.i.p. twice a day for two days) induced an increase in urine osmolality and significant increase in the Hyal-1 and Hyal-2 mRNA content in the medulla without changes in the cortex and papilla. The effect was more pronounced in Brattlboro rat kidney. These results demonstrate that, in control conditions, genes encoding Hyal-1 and Hyal-2 were expressed independently in all functional kidney zones in the both in normal Wistar and in vasopressin-defficient Brattlboro rats. Desmopressin (dDAVP) exerts a stimulating effect on Hyal-1 and Hyal-2 gene expression in the medulla.     

Long-Term Consumption of High-Fat Diet in Rats: Effects on Microglial and Astrocytic Morphology and Neuronal Nitric Oxide Synthase Expression

Obesity in humans is associated with cognitive decline and elevated risk of neurodegenerative diseases of old age. Variations of high-fat diet are often used to model these effects in animal studies. However, we previously reported improvements in markers of memory and learning, as well as larger hippocampi and higher metabolite concentrations in Wistar rats fed high-fat, high-carbohydrate diet (HFCD, 60 % energy from fat, 28 % from carbohydrates) for 1 year; this diet leads to mild ketonemia (Setkowicz et al. in PLoS One 10:e0139987, 2015). In the present study, we follow up on this cohort to assess glial morphology and expression of markers related to gliosis. Twenty-five male Wistar rats were kept on HFCD and twenty-five on normal chow. At 12 months of age, the animals were sacrificed and processed for immunohistochemical staining for astrocytic (glial fibrillary acidic protein), microglial (Iba1), and neuronal (neuronal nitric oxide synthetase, nNOS) markers in the hippocampus. We have found changes in immunopositive area fraction and cellular complexity, as studied by a simplified Sholl procedure. To our knowledge, this study is the first to apply this methodology to the study of glial cells in HFCD animals. GFAP and Iba1 immunoreactive area fraction in the hippocampi of HFCD-fed rats were decreased, while the mean number of intersections (an indirect measure of cell complexity) was decreased in GFAP-positive astrocytes, but not in Iba1-expressing microglia. At the same time, nNOS expression was lowered after HFCD in both the cortex and the hippocampus.     

Cloning and localization of rgpr85 encoding rat G-protein-coupled receptor

In an attempt to isolate genes involved in the brain development using ordered differential display PCR, we cloned rgpr85 which encodes rat G-protein-coupled receptor with high degree of identity to the amine-like neurotransmitter receptors. This gene was found to be localized at rat chromosome 4q21. In situ hybridization demonstrated that rgpr85 was predominantly expressed in the developing brain and spinal cord. Hybridization signal was especially abundant within the embryonic cortical plates where postmitotic cortical neurons are localized. In the cerebral cortex, the expression of rgpr85 was gradually decreased postnatally and became undetectable by P18. However, weak but significant expression of rgpr85 was maintained in the adult hippocampal formation, olfactory bulb, and cerebellum. Interestingly, rgpr85 expression was transiently induced in the adult hippocampal formation, piriform cortex, and amygdaloid complex by kainic acid (KA) treatment. Thus, dynamic regulation of rgpr85 expression suggests an importance of rgpr85-mediated signaling in the development of cerebral cortex and in the KA-induced responses in the adult brain.     

大鼠脑中5—羟色胺受体的检定及其在衰老时的变化

采用放射性配基结合分析法,对大鼠大脑皮质的5-HT受体作了检定,并观察了老年大鼠(36月龄)大脑皮质中该受体的变化。证实大鼠大脑皮质存在着丰富的、高亲和力和单一结合位点的5-HT受体。老年大鼠大脑皮质中5-HT受体的数目较成年大鼠(3月龄)明显减少,但亲和力无改变。应用荧光分光技术测定了成年和老年大鼠脑干和大脑皮质5-HT含量,证实老年大鼠上述两个脑区的5-HT含量均有降低。本研究的结果提示,老年大鼠中枢5-HT系统的功能减低,这一变化可能与老年期的一些表现如睡眠障碍、体温低、记忆力减退和易患精神疾病等有关。     

拉莫三嗪对GHB 致失神发作大鼠脑内HCN1、HCN2表达的影响

目的:观察拉莫三嗪对γ-羟丁酸(GHB)致失神发作大鼠脑电及脑内超极化激活环核苷酸门控阳离子通道(HCN)的亚型HCN1、HCN2表达变化的影响,探讨拉莫三嗪抗失神癫痫的可能作用机制。方法:健康成年雄性SD大鼠,随机分为空白对照组,模型组,拉莫三嗪治疗组(低剂量组为8 mg/(kg·d)、中剂量组为12 mg/(kg·d)、高剂量组为24 mg/(kg·d)),每组7只。空白对照组及模型组每日应用0.25%的甲基纤维素钠溶液灌胃,治疗组每日应用0.25%的甲基纤维素钠溶液配制的浓度为2 mg/mL的拉莫三嗪混悬液灌胃。手术埋置皮层脑电电极。腹腔注射GHB的前体γ-丁内酯(GBL)200 mg/kg制作大鼠失神发作模型,并监测脑电。免疫组化法检测皮层HCN1及丘脑HCN2的表达。结果:皮层脑电图拉莫三嗪治疗组比模型组失神发作的潜伏期延长,最高波幅降低(P0.05)。模型组皮质HCN1比空白对照组表达减少,而拉莫三嗪高、中剂量组皮质HCN1比模型组表达增加(P0.05)。模型组丘脑HCN2表达减少,与空白对照组及治疗组相比,差异有统计学意义。结论:拉莫三嗪可以改善GHB致失神发作模型脑电图的异常表现;拉莫三嗪抗失神癫痫作用可能与调节HCN表达有关。