甲状腺激素反应蛋白1基因-一个新的鼠脑甲状腺激素反应基因的克隆、表达分布及其功能预测

目的 克隆新的大鼠脑甲状腺激素反应基因，研究其表达特征并初步预测其功能。方法 建立先天性甲状腺功能减退(甲减)新生大鼠模型，用荧光标记的差异显示PCR(DD-PCR)技术、亚克隆、测序及相似性检索获得未知的大鼠脑甲状腺激素反应基因的cDNA片段。采用电子克隆、RT-PCR、测序并克隆其cDNA全长。经Northem印迹法证实其表达受到甲状腺激素的调节，并用半定量RT-PCR方法和生物信息学技术观察其分布转录水平和预测其功能。结果克隆了甲状腺激素反应蛋白1(thyroid hormoneresponse protein-1，TRP-1)基因全长cDNA，共973bp(基因登录号为AF348365)，在甲减新生大鼠的脑中，该基因的转录增强。该基因表达广泛，以脑组织中的表达水平最高；其在大鼠不同脑区的表达含量亦不同，以嗅脑最高。该基因的编码产物具有一些重要的结构域和功能基序。结论 TRP-1基因是新发现的甲状腺激素反应基因，它在正常的脑发育中可能具有重要的作用，其异常表达可能是甲减性脑发育障碍发生的分子机制之一。     

甲状腺激素对成骨样MG-63细胞甲状腺激素受体不同亚型表达的影响

目的 检测人类成骨样细胞MG-63中甲状腺激素受体(TR)的表达情况以及甲状腺激素(T3或T4)对其的影响.方法 用实时荧光定量PCR法对MG-63细胞中TR的主要4个亚型,α1、α1、β1、β2的mRNA表达进行了测定.结果 TRα1 mRNA的表达水平最高,为10.70±0.45;TRβ1仅及其半,为5.75±0.10;TRβ2不低,相当于TRβ1的60%,为3.34±0.08;TRα2的表达极低,仅为(3.66±0.59)×10-2.分别加入不同浓度的T3或T4,仅TRα1和TRα2对T3表现明显的降调节,且TRα1和TRα2的表达与相应T3浓度之间负相关,其他亚型无显著性变化,也无相关关系;同样浓度范围的T4作用各亚型,表达均无显著性变化.结论 在骨细胞发育分化中TRα1起关键作用.

Abstract：

Objective To examine the expression of thyroid hormone receptor isoforms (TR α1, α2, β1, and β2) in human osteoblast-like cell line MG-63 at the mRNA level and the effect of thyroid hormone (T3 or T4 ) on the expression. Methods Realtime quantitative PCR was performed. Results The expression of TRα1 mRNA was the highest, that was 10. 70± 0.45, TRβ1 was 5.75 ± 0. 10, TRβ2 was 3.34 ± 0. 08, and TRα2 was very low, only (3.66 ±0. 59) × 10-2. Only the expression of TRαl and TRα2 mRNA was down regulated significantly by the treatment of 10-10 ～ 10-6 mol/L T3, and there was a negative correlation between the expression of TRα1 or TRα2 mRNA and the concentration of T3. Conclusion TRα1 plays a primary role in mediating the effects of thyroid hormones in skeletal development.     

碘过量对大鼠仔鼠垂体促甲状腺激素细胞的影响

目的探讨碘过量对仔鼠垂体TSH细胞的影响。方法将断乳1个月的W istar大鼠,雌雄各半随机分为4组:NI组、10H I组、50H I组和100H I。饲养3个月的雌雄大鼠1∶1合笼交配产生仔鼠,断乳后的仔鼠喂养同上述大鼠。测定出生60日龄仔鼠的甲状腺和垂体的相对重量及垂体TSH细胞的体密度和面数密度。结果50H I组、100H I组仔鼠的体密度和面数密度明显高于NI组和10H I组,但垂体和甲状腺的相对重量与NI组相比无明显变化。结论50H I组、100H I组不会引起仔鼠甲状腺肿,甲状腺对碘过量有较强的适应性和耐受性。     

甲状腺激素缺乏对发育期鼠脑蛋白激酶C活性的影响

目的 研究甲状腺激纱缺乏对发育期大鼠脑蛋白激素Ｃ（ＰＫＣ）活性的影响,以探讨甲状腺激素调节脑发育的机制。方法 以丙基硫氧嘧啶溶液灌胃造成大鼠围生期甲状模型,部分甲状仔嫌每日腹腔注射Ｔ４２μｇ／１００ｇ体重。收集５天、１５天和２５天的正常、甲减及Ｔ４－替代治疗仔鼠脑组织、测定脑组织胞液和膜ＰＫＣ活性。结果 生后５天、１５天和２５天的甲减仔鼠脑胞液ＰＫＣ活性组织,测定脑组织胞液和膜ＰＫＣ活性。结果 和     

大鼠脑单胺氧化酶对甲状腺激素反应的实验观察

目的 探讨甲状腺激素对甲状腺功能低下（甲低）鼠脑单胺氧化酶（MAO）活性的影响和保护作用。方法 用1%NaClO4诱发大鼠甲状腺功能低下,实验组大鼠分别注射T3、T4,观察脑MAO活性变化,同时观察生后不同时期甲低仔鼠MAO活性。结果 甲低大鼠脑MAO活性增强,20日龄仔鼠MAO活性增加的百分比高于成熟鼠。注射T4脑MAO活性恢复,脑MAO活性与血清T4呈负相关。结论 大鼠甲低时脑MAO活性变化反映了MAO在维持大脑发育和功能上起重要作用,T4对脑MAO活性有保持作用。     

甲状腺功能减退对新生早期大鼠各脑区甲状腺激素受体mRNA表达的影响

目的探讨甲状腺功能减退（甲减）对新生早期大鼠各脑区甲状腺激素受体（TR）mRNA表达的影响。方法建立甲减Wistar大鼠动物模型，分别于仔鼠0、14、21、45d，用实时荧光定量PCR方法检测大脑、小脑、脑干和海马TR mRNA的表达。结果与对照组相比，各时间点甲减仔鼠各脑区TRα1 mRNA的表达量呈总体下调趋势，而甲减0d仔鼠大脑、小脑、脑干TRα2 mRNA表达量明显增高（t=8．18、6．23、3．68，P〈0．01），且45d仔鼠各脑区TRα2 mRNA表达量仍高于对照组（t大脑=5．50、t小脑=5．46、t脑干=4．10、t海马=11．83，P〈0．01），TRα1、TRα2 mRNA表达峰（21d）均延迟于对照组（14d）出现。甲减仔鼠TRβ1 mRNA表达变化趋势与对照组相一致，但45d仔鼠各脑区TRβ1 mRNA表达量均低于对照组（t大脑=4．64、t小脑=2．73、t脑干=3．90、t海马=5．07，P〈0．01或〈0．05）。结论甲减时甲状腺激素受体mRNA表达峰值的延迟出现以及异常的表达变化与克汀病脑损伤机制密切相关。     

甲状腺激素对成年脑的作用

甲状腺激素对胚胎及新生儿期脑的发育具有关键作用，但对成年后大脑的作用存在争议。近年来甲状腺激素对成年和老年期脑的作用越来越受到人们的关注，现就此方面的研究现状综述如下。[第一段]     

甲状腺激素受体基因在体外培养低碘鼠脑神经细胞中的表达

应用ＤＮＡ－ＲＮＡ斑点杂交方法研究了低碘鼠模型第１５－１８日龄胎鼠脑神经细胞原代培养物甲状腺激素受体（ＴＨＲ）ｃ－ｅｒｂＡα ｍＲＮＡ的表达。结果显示大脑神经细胞在０天时已可检测到ＴＨＲｍＲＮＡ的表达，培养第５天时表达水平略有增高，培养第１０天时表达量大约可达８０ｐｇ左右，在培养第１０－２０天之间ＴＨＲｍＲＮＡ表达水平没有明显变化。在培养期间小脑神经细胞ＴＨＲｍＲＮＡ的表达水平较低，并且没有明显的     

内皮素和心钠素调节TRH受体基因的表达

目的研究神经肽内皮素（ＥＴ）和心钠素（ＡＮＰ）在细胞培养及体内实验中对促甲状腺激素释放激素受体（ＴＲＨＲ）基因表达的影响。方法Ｎｏｒｔｈｅｒｎ印迹和Ｓ１核酸酶保护实验检测ＴＲＨＲｍＲＮＡ量，新生肽链试验测定ＴＲＨＲ基因的转录。结果ＥＴ３２ｈ即增加胶质细胞ＴＲＨＲｍＲＮＡ量，６～８ｈ达高峰，增加３倍以上（３．０±０．５２）；相反，ＡＮＰ２ｈ即降低ＴＲＨＲｍＲＮＡ，６～８ｈ达最低值，并逆转６０～８０％因血浆所增高的ＴＲＨＲｍＲＮＡ。ＥＴ３在６ｈ时增加ＴＲＨＲ基因的转录达２倍（ｎｕｃｌｅａｒｒｕｎｏｎ试验），而ＡＮＰ对ＴＲＨＲ基因的转录无明显变化。体内实验结果显示ＥＴ３和ＡＮＰ对ＴＲＨＲｍＲＮＡ影响与体外实验相似。结论ＥＴ３可能通过调节ＴＲＨＲ基因的表达而影响ＴＲＨＲｍＲＮＡ，而ＡＮＰ则可能影响ＴＲＨＲｍＲＮＡ的降解而降低ｍＲＮＡ的水平。     

甲状腺素对幼鼠脑TNF-α和TNFR1基因表达的影响

目的研究甲状腺素(T4)对幼鼠脑TNF-α和TNFR1基因表达的影响.方法采用丙基硫氧嘧啶灌胃制备甲状腺功能低下(甲低)孕鼠模型,出生后小鼠为先天性甲低鼠,另一组出生后即日起用腹腔注射T4,剂量为20 μg/(kg*d)(替代组),采用逆转录-聚合酶链反应检测出生后1,5,10 d幼鼠TNF-α和TNFR1基因的表达.结果在出生后第1 d甲低幼鼠脑TNF-α和TNFR1基因表达最高,随着鼠龄增长,表达降低;正常出生后第1 d幼鼠及替代组TNF-α和TNFR1基因表达较甲低组低.结论甲状腺素对TNF-α和TNFR1基因表达的影响在脑细胞凋亡中有重要的作用.     

甲状腺素对仔鼠脑NAPOR基因表达的影响

目的研究甲状腺素对仔鼠脑成纤维母细胞瘤凋亡相关的RNA结合蛋白(neuroblastoma apoptosis-related RNA-binding protein,NAPOR)基因表达的影响。方法以丙基硫氧嘧啶灌胃复制孕鼠甲状腺功能低下模型,采用逆转录-聚合酶链反应检测仔鼠出生后第1、5、10天仔鼠皮层NAPOR的表达,年龄匹配的正常仔鼠为对照组。结果NAPOR在甲状腺功能低下孕鼠所产仔鼠表达均高于正常对照组;在出生后一段时期内,随着鼠龄增长表达降低。结论转录后水平的调节机制可能参与甲状腺素对脑基因表达的影响。     

Cellular-resolution gene expression profiling in the neonatal marmoset brain reveals dynamic species- and region-specific differences

Precise spatiotemporal control of gene expression in the developing brain is critical for neural circuit formation, and comprehensive expression mapping in the developing primate brain is crucial to understand brain function in health and disease. Here, we developed an unbiased, automated, large-scale, cellular-resolution in situ hybridization (ISH)–based gene expression profiling system (GePS) and companion analysis to reveal gene expression patterns in the neonatal New World marmoset cortex, thalamus, and striatum that are distinct from those in mice. Gene-ontology analysis of marmoset-specific genes revealed associations with catalytic activity in the visual cortex and neuropsychiatric disorders in the thalamus. Cortically expressed genes with clear area boundaries were used in a three-dimensional cortical surface mapping algorithm to delineate higher-order cortical areas not evident in two-dimensional ISH data. GePS provides a powerful platform to elucidate the molecular mechanisms underlying primate neurobiology and developmental psychiatric and neurological disorders.

The mammalian brain contains many functionally distinct regions that are extensively interconnected, enabling rapid and accurate information processing. Each region contains diverse cell types that differ in their molecular, morphological, electrophysiological, and functional characteristics (1, 2). Advanced neuroscience technologies, which are generally optimized and extensively applied in mice, provide complicated approaches to analyze these characteristics and model the genetic basis of this regional- and cell-type diversity. However, clear cognitive differences arise from interspecies differences in these key characteristics, and most contemporary technologies are difficult to apply in species whose study involves extensive ethical, practical, and experimental impediments. The ability to compare gene expression patterns between primates and model species such as mice is crucial for understanding the human brain (3, 4).Recent developments in technologies, such as microarray, single-cell RNA-sequencing (RNA-seq), and Drop-seq, have allowed for high-resolution genome-wide expression analysis in specific regions of the brain or in thousands of individual cells simultaneously (5–10). However, these methods provide limited anatomical information and no morphological observations. Slide-seq solves these problems by transferring RNA from tissue sections onto a surface covered in DNA-barcoded beads, allowing the preservation of positional information (11). However, the unbiased nature of next-generation sequencing technologies may hinder the molecular identification of specific cell types or their direct analysis. In situ hybridization (ISH) databases of humans and nonhuman primates (NHPs) have revealed brain gene expression profiles at cellular resolution, while preserving key morphological and anatomical characteristics, ultimately providing important information about the genetic conservation of analogous brain regions (12). However, these studies are highly resource intensive, requiring substantial time and cost. Consequently, such efforts have been limited, focusing on a few brain regions in limited species (13). Thus, interspecific comparison of brain-cell heterogeneity has not been possible.Here, we describe the development and implementation of an unbiased gene expression profiling system (GePS) for use with the Marmoset Gene Atlas (MGA) (https://gene-atlas.brainminds.riken.jp/) (14), which is adaptable for use with other atlases. The system, developed as a part of “Brain Mapping by Integrated Neurotechnologies for Disease Studies (Brain/MINDS),” a brain mapping program in Japan (15, 16), enables systematic and automatic analysis of gene expression across brain regions. Brain/MINDS aims to develop knowledge-based tools to explore primate-specific brain structure, function, and connectivity in health and disease. Specifically, we analyzed the expression patterns of identified risk genes for psychiatric disorders in the neonatal marmoset brain. There are >2,000 gene expression profiles for the neonatal common-marmoset brain in the MGA. Although each ISH section is complemented by an adjacent neuroanatomically stained section (visualizing the Nissl substance), enabling users to compare gene expression against a brain atlas, mapping gene expression patterns over the entire brain is both time and labor intensive. A large mouse ISH database, the Allen Brain Atlas (ABA; http://developingmouse.brain-map.org/), demonstrated that an anatomic gene expression atlas (AGEA) can characterize local gene expression in the mouse brain without prior knowledge of classical anatomy (17). To enhance the MGA in this manner, we developed GePS to systematically and automatically identify gene expression patterns in specific regions of the neonatal marmoset brain.GePS, including the gene expression comparison system and cortical surface projection mapping function, is publicly available as an open resource on our website (https://gene-atlas.brainminds.riken.jp/). It provides primate-specific whole brain–gene expression patterns and will be a valuable platform in the field of primate neuroscience, helping reveal primate-specific brain functions and connections and providing insights into brain evolution and pathology.     

Variable regulation by insulin of insulin gene expression in HIT-T15 cells

Summary Glucose and insulin are generally considered to express opposite effects on insulin synthesis and secretion from pancreatic islets. For the most part this generalization has arisen from short-term experiments. Our studies focused on the chronic, long-term effects of variable insulin concentrations on insulin gene expression and secretion in cultures of HIT-T15 cells. From passage 70, HIT cells were split and passed weekly for 25 weeks in media containing either (A) 11.1 mmol/l glucose with no insulin added; (B) 11.1 mmol/l glucose with insulin added to maintain a level of approximately 4,000 U/ml; (C) 0.8 mmol/l glucose with no insulin added; (D) 0.8 mmol/l glucose with insulin added to maintain a level of approximately 4,000 U/ml; and (E) 0.8 mmol/l glucose with progressively less insulin added over time to mimic the gradual decrease in media insulin levels found in condition A. Our data indicate that during chronic passing of HIT cells, addition of exogenous insulin led to preserved levels of insulin mRNA, insulin content and insulin secretion in cells cultured in media containing 11.1 mmol/l glucose concentration. However, in media containing 0.8 mmol/l glucose concentration, addition of insulin diminished the levels of insulin mRNA, insulin content and insulin secretion. Nonetheless, in all cases exogenously added insulin sustained greater levels of insulin mRNA, insulin content and insulin secretion than the instance wherein media containing a high concentration of glucose only was used. These results indicate that short-term experiments assessing the effects of insulin on beta-cell function do not necessarily apply to chronic, more long-term experiments in which insulin can have variable time- and concentration-dependent effects on insulin gene expression and secretion.Abbreviations HIT Hamster insulin-secreting tumour     

Hormonal regulation of cAMP-independent protein phosphokinase activities: thyroxine and cortisol control of enzymes from rat liver cytosol.

Thyroxine control of cAMP-independent histone and casein phosphokinase activities was studied in thyroidectomized rats treated with thyroxine. All activities were evaluated in the presence of a thermostable inhibitor of cAMP-dependent enzymes. Cytosol enzymes can be resolved by sucrose gradient ultracentrifugation into three peaks of histone kinase activity (3.2S, 5S and 7.2S) and two peaks of casein kinases (3.6S and 7.1S). Neither thyroidectomy nor subsequent treatment of operated animals with thyroxine modifies the total histone kinase activity estimated, either in total cytosol or after its fractionation by the sucrose gradient ultracentrifugation. The activity ratios of different peaks were, however, changed. Casein kinase activity was significantly decreased after thyroidectomy (about 50%). Subsequent treatment with thyroxine restored this activity to its initial value. Sucrose gradient ultracentrifugation analysis showed that thyroxine action on the casein kinase activity is very specific. Only molecules that sediment in the 9S region were significantly stimulated by the hormone. Cortisol action on the casein kinase activity was studied in adrenalectomized animals treated with hormone for 24 h. Cortisol decreases the total casein kinase activity by about 30%. Sucrose gradient ultracentrifugation analysis showed that the population of molecules sedimenting at about 9S was the most sensitive to cortisol. The above data show that both thyroxine and cortisol control, in a selective way, the activities of cAMP-independent protein kinases. The same kinase molecules can be under double control by two different hormones that have opposite effects.     

Divergent and nonuniform gene expression patterns in mouse brain

Considerable progress has been made in understanding variations in gene sequence and expression level associated with phenotype, yet how genetic diversity translates into complex phenotypic differences remains poorly understood. Here, we examine the relationship between genetic background and spatial patterns of gene expression across seven strains of mice, providing the most extensive cellular-resolution comparative analysis of gene expression in the mammalian brain to date. Using comprehensive brainwide anatomic coverage (more than 200 brain regions), we applied in situ hybridization to analyze the spatial expression patterns of 49 genes encoding well-known pharmaceutical drug targets. Remarkably, over 50% of the genes examined showed interstrain expression variation. In addition, the variability was nonuniformly distributed across strain and neuroanatomic region, suggesting certain organizing principles. First, the degree of expression variance among strains mirrors genealogic relationships. Second, expression pattern differences were concentrated in higher-order brain regions such as the cortex and hippocampus. Divergence in gene expression patterns across the brain could contribute significantly to variations in behavior and responses to neuroactive drugs in laboratory mouse strains and may help to explain individual differences in human responsiveness to neuroactive drugs.     

应用基因芯片研究肝细胞癌组织差异基因表达谱

目的：应用基因芯片技术研究原发性肝细胞癌组织中的差异基因表达谱改变，以寻找肝细胞癌相关基因。方法：抽提正常肝组织和肝癌组织中的mRNA来制备探针，经杂交、洗涤后，通过计算机扫描分析正常肝组织和肝癌组织基因表达谱的差异情况。结果：在10000个候选基因中，筛选出102条差异表达基因，表达上调的有42条，表达下调的有60条。未知基因12条。结论：基于DNA微阵矩技术的肿瘤基因表达谱分析能够高通量筛选与肝癌发生发展相关的基因。     

大鼠AQP4基因表达载体的构建及在体内表达的观察

目的　克隆大鼠AQP4基因并构建其融合蛋白表达载体 ,以便载体在体内进行AQP4基因干预。方法　采用逆转录构建大鼠脑cDNA文库、聚合酶链反应 (PCR)扩增大鼠AQP4基因片断 ,选用绿色荧光蛋白 (GFP)为报告基因 ,将其构建到pcDNA3.1 Zeo(+)真核表达质粒中 ,同时在AQP4、GFP基因之间插入IRES片段 ,以构建AQP4 GFP融合蛋白。在脑立体定位仪下将经预先处理过的AQP4 GFP表达质粒注射到大鼠脑内 ,荧光显微镜下观测基因表达 ,免疫组织化学检测脑内AQP4表达水平的变化。结果　 (1)扩增出长度为 993bp的片段 ,为AQP4序列的全段 ,测序结果与基因Bank(U14 0 0 7)上报道的大鼠AQP4M1型基因序列相同。 (2 )注射实验质粒和对照质粒后 12h于注射点局部均可观察到GFP阳性细胞 ,高峰出现在 2 4h ,至 72h荧光明显减弱。转染实验质粒 2 4h后可明显提高脑内AQP4表达的水平。结论　克隆的大鼠AQP4基因和构建的AQP4表达质粒 ,在体内获得表达 ,该质粒可以用于干预体内AQP4基因表达的相关研究。