甲状腺激素反应蛋白1的原核生物表达

目的 应用基因工程技术表达甲状腺激素反应蛋白1(TRP-1)。方法 应用RT-PCR方法，从新生大鼠脑组织RNA中扩增编码TRP-1的cDNA片段，构建表达型重组质粒，经DNA序列分析确证后，在大肠杆菌中表达，以Western印迹来初步鉴定是否表达了目的融合蛋白，用亲和层析纯化融合目的蛋白，并以SDS-PAGE电泳测定其相对分子质量。结果 所获特异PCR产物正确地重组入Pinpoint Xa-1表达载体中。Western印迹表明经异丙基硫代半乳糖苷诱导的原核细胞表达产生了目的融合蛋白，亲和层析得到了纯度较高、相对分子质量约23400的融合目的蛋白。结论 应用原核细胞表达体系成功地表达了TRP-1融合蛋白，为进一步研究其在脑发育中的所起的作用以及该蛋白的其它功能提供了可能。     

甲状腺激素调节幼年大鼠海马Goα及Gsα基因表达的研究

甲状腺激素(ＴＨ)可直接或间接调控多种脑结构基因的表达〔1〕,但ＴＨ和脑功能相关基因,尤其信号蛋白编码基因的调节关系,至今仍研究较少。近来,我们的系列研究发现围生期甲减大鼠对下丘脑某些核团Ｇｏ蛋白α亚基(Ｇｏα)基因的表达具有显著的上调作用〔2〕。考虑到海马结构是参与学习、记忆等认知活动的重要中枢部位,而也是广泛表达Ｇｏα的脑区之一〔3〕,本研究进一步观察ＴＨ对发育期大鼠海马及齿状回Ｇｏα基因表达的调节作用,并对照研究ＴＨ对该脑区Ｇｓ蛋白α亚基(Ｇｓα)基因表达的影响。一、材料与方法1.材料:孕Ｗｉｓｔａｒ大鼠,Ｆ…     

甲状腺激素对脑发育的重要影响

甲状腺激素(TH)在哺乳类动物脑发育中具有重要的作用。孕母亚临床甲状腺功能减退症(甲减)可造成胎儿神经发育受损，对孕妇作甲减筛查是值得推荐的做法。脑TH反应基因的鉴定及功能研究有助于阐明TH调节脑发育的分子机制。     

甲状腺激素对新生期大鼠下丘脑Go蛋白α亚单位的影响

目的　研究新生期Ｗｉｓｔａｒ 大鼠下丘脑Ｇｏ 蛋白α亚单位（Ｇｏα） 的分布及甲状腺激素对其表达的影响。方法　采用地高辛标记寡核苷酸探针原位杂交技术,对新生期正常及甲减Ｗｉｓｔａｒ 大鼠下丘脑ＧｏαｍＲＮＡ 的表达状况进行研究。结果　（１）ＧｏαｍＲＮＡ 主要分布于新生期Ｗｉｓｔａｒ 大鼠下丘脑的弓状核（ＡＲ）、腹内侧核（ＶＭＨ）等部位;（２）２１ 日龄组大鼠下丘脑诸核团ＧｏαｍＲＮＡ 的转录水平明显低于１４ 日龄组;（３） 甲状腺功能低下可引起１４ 日及２１ 日龄大鼠下丘脑ＡＲ、ＶＭＨ 核团ＧｏαｍＲＮＡ水平显著升高。结论　在大鼠下丘脑的发育过程中,信号蛋白Ｇｏα基因呈现特征性分布及表达,甲状腺激素对该基因具有转录下调作用。     

甲状腺激素对大鼠小脑,海马,嗅脑神经元型一氧化氮合酶基因表达的调节

目的　观察 15日龄大鼠小脑 ,海马 ,嗅脑一氧化氮 (NO)含量 ,一氧化氮合酶 (NOS)活性的变化及甲状腺激素对上述部位神经元型一氧化氮合酶 (nNOS)基因表达的调节。方法　采用丙基硫氧嘧啶 (PTU)给孕母鼠灌胃造成仔鼠甲减动物模型 ;采用NO ,NOS生化测定法及nNOSmRNA半定量逆转录聚合酶链反应 (RT PCR)法。结果　无论正常组还是甲减组 ,NO含量 ,NOS活性及nNOSmRNA转录均以小脑为最高 ,嗅脑次之 ,海马最低 (P <0 .0 5 ) ;正常组NO含量 ,NOS活性nNOSmRNA转录均高于甲减组 (P <0 .0 1)。结论　提示甲状腺激素对nNOS基因表达有上调作用 ,NO信号系统可能参与大鼠小脑 ,海马 ,嗅脑等区甲状腺激素缺乏所造成的脑损害过程。     

甲状腺激素调节仔鼠脑基因差异表达的研究

目的　研究甲状腺激素调节仔鼠脑基因的分子机制。方法　丙基硫氧嘧啶灌胃制备孕鼠甲状腺功能减退模型 ,部分胎鼠出生后予以 T4 替代治疗 ,采用消减抑制杂交技术构建甲状腺激素调节仔鼠脑基因的差异表达文库 ,对部分重组克隆进行序列分析。结果　随机测序 10个表达序列标签 ,同源性分析表明 5个表达序列标签未发现明确的同源物 ,其它表达序列标签与 CDC10 ,酸性核糖体蛋白 P0 ,actin等有较高同源性。结论　甲状腺激素调节仔鼠脑基因的差异表达谱的获得对阐明甲状腺激素对脑发育的分子机理有十分重要的意义     

肝细胞癌高表达基因片段P02的全长RACE扩增和序列分析

目的扩增肝细胞癌高表达基因基因P0 2的全长cDNA序列,并利用生物信息学知识对其进行分析,为进一步研究该基因在肝细胞癌发生、发展中的作用奠定基础。方法运用SMARTRACE(RapidAmplificationofcDNAEnds)技术扩增P0 2的5’和3’末端cDNA ,对RACEPCR产物作TA克隆,碱裂解法抽提阳性克隆质粒,酶切和PCR验证目的片断的插入,Sanger双脱氧链中止法测序,利用BLAST、基因探索者等生物学软件对序列进行分析、拼接,获得全长cDNA ,利用在线预测软件预测该基因的生物学功能。结果获得865bp的全长cDNA序列,其开放读码框长172个氨基酸,与TPT1基因高度同源,是一个生理功能尚未完全明了、与生长相关的蛋白质,蛋白质结构和功能预测提示该基因产物可能是一个位于细胞膜的与生长相关的裂解酶。结论在肝细胞癌组织中成功克隆扩增了长865bp的P0 2全长cDNA序列,为进一步功能研究奠定基础。     

缺碘性甲状腺功能低下大鼠脑中甲状腺激素受体基因的表达

目的已知缺碘性甲状腺功能低下(甲低)大鼠脑中甲状腺激素受体(T3受体)的最大结合容量MBC(代表受体浓度)升高.本文是在分子水平探讨此种升高是否发生在转录水平,即是否受体的mRNA增加.方法从缺碘饲养下(其血中T4水平下降)的1日龄和20日龄仔鼠的大脑中提取总RNA,以本室克隆的大鼠α1型T3受体cDNA标记后作为探针,经Northern印渍分析;并以同一标本中18sRNA为参比.经光密度扫描,求出受体mRNA与18sRNA的比值,然后与同龄正常鼠脑的数值进行比较.结果 1日龄甲低α1型T3受体mRNA的表达量(以T3受体值/18sRNA比值表示)比正常者显著升高,即分别为0.54±0.06和0 41±0.08;同样20日龄的比值分别为2.47±1.39和1.15±0.31.可见虽然脑中T3受体基因的表达随发育而增加,但在20 d以前甲低者显著高于正常;与我们先前用Scatchard动力分析法所测T3受体蛋白浓度(MBC)因缺碘而致甲低大鼠脑中显著高于正常者的结果相符.受体表达的增加,在基因表达的早期-转录水平即已发生.结论大鼠脑中T3受体基因表达虽可随着发育而与日俱增,但缺碘大鼠总比正常者表达水平升高,至少在20 d以内如此.     

日本血吸虫TEGT基因的cDNA克隆和生物信息学分析

目的克隆日本血吸虫新基因TEGT(Sj.TEGT)的全长序列。方法根据编码日本血吸虫大陆株的EST基因片段的序列,设计5’RACE的系列引物,扩增Sj.TEGT的5’端,测序后预测该基因的全长ORF;再以此设计引物,以日本血吸虫中国大陆株成虫mRNA为模板,用RT-PCR方法扩增得到Sj.TEGT的全长cDNA。结果从日本血吸虫中国大陆株成虫mRNA中克隆到1个TEGT相关基因,命名为Sj.TEGT基因。其编码的蛋白的分子质量大约为30ku;对其编码的氨基酸序列的分析显示其编码蛋白有6个跨膜螺旋区。结论Sj.TEGT是一个新基因,有必要对其功能进行深入研究。     

Two-dimensional complementary deoxyribonucleic acid electrophoresis revealing up-regulated human epididymal protein-1 and down-regulated CL-100 in thyroid papillary carcinoma

Two-dimensional cDNA electrophoresis was used to analyze gene expressions in papillary carcinoma and normal tissue of thyroid glands. Pooled thyroid tissues were used to extract mRNA. Complementary DNAs, synthesized with NotI anchor primers, were digested with three restriction enzymes, NotI, EcoRV, and PvuII. The protruding NotI ends were filled in with (32)P deoxynucleotide triphosphates, and the radiolabeled cDNA fragments were separated in two dimensions. Approximately 500 cDNA fragments were visualized as discrete spots without probes. A total of 20 spots, 9 up-regulated and 11 down-regulated cDNAs in papillary carcinoma, were selected and cloned for sequencing. This experiment lent itself to a novel discovery of up-regulated human epididymal protein 1 (HE-1) and down-regulated CL-100 genes in thyroid papillary carcinomas confirmed by Northern blot analysis. Immunohistochemical stains showed abundant HE-1 protein in the papillary carcinoma, whereas little or no HE-1 protein was detected in other types of thyroid cancers and normal thyroid tissues. The restricted localization of HE-1 protein to the portions of papillary projections suggests an involvement of HE-1 protein for forming papillary shape. Our study showed that two-dimensional cDNA electrophoresis is a useful method of detecting differentially expressed genes in human diseases as demonstrated for HE-1 and CL-100 in papillary carcinoma.     

Increased expression of mitochondrial genes in human alcoholic brain revealed by differential display

Polymerase chain reaction (PCR)-based differential display was used to screen for alterations in gene expression in the mesolimbic system of the human alcoholic brain. Total RNA was extracted from the nucleus accumbens of five alcoholic and five control brains. A selected subpopulation of mRNA was reverse-transcribed to cDNA and amplified by PCR. A differentially expressed cDNA fragment was recovered, cloned, and sequenced. Full sequence analysis of this 467 bp fragment revealed 98.2% homology with the human mitochondrial 12S rRNA gene. Dot-blot analysis showed increased expression of this gene in nucleus accumbens and hippocampus, but not in the superior frontal cortex, primary motor cortex, caudate, and pallidus/putamen in a total of eight human alcoholic brains, compared with seven control brains. A similar increased expression was observed by dot-blot analysis, using RNA from the cerebral cortex of rats chronically treated with alcohol vapor. Hybridization of a 16S rRNA oligonucleotide probe indicated that the expression of both rRNAs genes was significantly increased in nucleus accumbens. These results indicate that chronic alcohol consumption induces alteration in expression of mitochondrial genes in selected brain regions. The altered gene expression may reflect mitochondrial dysfunction in the alcohol-affected brain.     

Tensin3 is a novel thyroid-specific gene

Thyroid-specific genes, such as thyroid peroxidase, thyroglobulin, Na+/I- symporter and thyroid-stimulating hormone receptor, play fundamental roles in thyroid function and relate to many pathological conditions. Using sequence specific-differential display, we detected three genes that showed higher expression levels in normal thyroid tissues than in thyroid tumor tissues. After subcloning and sequencing analysis, one of the genes was revealed to be tensin3. The expression level of tensin3 was examined with real-time quantitative PCR analysis. Its expression levels were more depressed in thyroid tumor tissues than in normal thyroid tissues. The decrease was even more evident in two anaplastic carcinomas. High and moderate levels of tensin3 mRNA expression were observed in the thyroid and placenta respectively. Tensin3 mRNA was expressed only in low levels in other tissues, such as the brain, heart, lung, liver, pancreas, kidney, skeletal muscle, white blood cells and prostate. These results show that tensin3 is a novel thyroid-specific gene and further investigations may reveal its relation to thyroid function or thyroid disease.     

A novel seizure-induced synaptotagmin gene identified by differential display

Systemic administration of kainic acid, a cyclic analogue of glutamate, produces many of the clinical features of human temporal lobe epilepsy and status epilepticus in rats, including the induction of motor convulsions and the degeneration of neurons in the hippocampus and piriform cortex. Differential display PCR was used to identify mRNAs that are differentially expressed between degenerating and nondegenerating tissues in the brain after kainic acid-induced seizure activity. A novel cDNA fragment expressed in the degenerating hippocampus and piriform cortex, but not in the nondegenerating parietal cortex, was identified, cloned, and sequenced. This novel cDNA fragment identified a new member of the synaptotagmin gene family that is rapidly and transiently induced in response to seizure activity. Differential expression of this synaptotagmin gene, syt X, was confirmed by Northern blot analysis and in situ hybridization. This novel, inducible synaptotagmin gene may provide a direct link between seizure-induced neuronal gene expression and subsequent modulation of synaptic structure and function.     

Genomic organization of mouse ZAKI-4 gene that encodes ZAKI-4 alpha and beta isoforms, endogenous calcineurin inhibitors, and changes in the expression of these isoforms by thyroid hormone in adult mouse brain and heart

OBJECTIVE: ZAKI-4 was identified as a thyroid hormone-responsive gene in cultured human fibroblasts. A single ZAKI-4 gene encodes two isoforms, ZAKI-4 alpha and beta, both inhibiting calcineurin activity. ZAKI-4 alpha and beta differ at their N termini, and show distinct distribution profiles in human tissues. The aim of this study was to elucidate the organization of the mouse ZAKI-4 gene and to determine the effect of thyroid hormone on the expression of ZAKI-4 isoforms in vivo. DESIGN: We cloned mouse homologues of human ZAKI-4 alpha and beta cDNA. Fluorescence in situ hybridization and bioinformatics analysis were employed to determine the gene organization. The effect of thyroid hormone on the expression of ZAKI-4 isoforms in mouse brain and heart was also studied. METHODS: Total RNA extracted from mouse cerebellum was used to clone ZAKI-4 alpha and beta cDNAs by RT-PCR followed by rapid amplification of cDNA ends. Mice were rendered hypothyroid by feeding a low iodine diet supplemented with propylthiouracil for 2 weeks. In one group (hyperthyroid) L-T(3) was injected i.p. for the last 4 days whereas another group (hypothyroid) received vehicle only. Non-treated mice were controls. RESULTS AND CONCLUSION: Mouse ZAKI-4 alpha and beta cDNAs were highly homologous to the human isoforms. The gene was mapped on chromosome 17qC, syntenic to human chromosome 6 where the human ZAKI-4 gene is located. As observed in human, ZAKI-4 alpha mRNA was expressed only in brain whereas beta mRNA was distributed in other tissues as well, such as heart and skeletal muscle. ZAKI-4 alpha mRNA was lower in the cerebral cortex of hypothyroid mice. Injection of L-T(3) caused an increase in ZAKI-4 beta mRNA in heart; however, expression of neither ZAKI-4 alpha nor beta mRNA was influenced by thyroid status in other tissues. These results indicate that expression of ZAKI-4 alpha and beta isoforms is regulated by thyroid hormone in vivo, and the regulation is isoform- and tissue-specific.     

日本血吸虫亲环素A和乳酸脱氢酶编码基因的克隆及序列分析

目的　识别和克隆日本血吸虫新基因。方法　对本实验室获得的EST进行同源性分析 ,识别血吸虫新基因 ;根据EST设计引物 ,用锚着PCR法从cDNA文库中扩增出新基因全长cDNA ,并将其克隆入原核表达载体 pGEX - 4T - 1,用生物信息学技术对获得的编码基因进行结构与功能的分析。结果　EST同源性分析得到一个完整编码基因 ,开放阅读框(ORF) 4 95bp ,编码 16 4个氨基酸 ;锚着PCR法获得另一EST的 3’端所缺序列 ,得到完整编码阅读框 ,其ORF 999bp ,编码 332个氨基酸。利用生物信息学技术确定它们分别为日本血吸虫亲环素A(CyPA)和乳酸脱氢酶 (LDH)的编码基因。重组质粒经双酶切及测序鉴定证明日本血吸虫CyPA和LDH原核表达质粒构建成功。 结论　克隆到日本血吸虫亲环素A和乳酸脱氢酶编码基因的全长cDNA ,为进一步的功能研究打下了基础     

人甲状腺乳头状癌高表达基因片段筛选与克隆

应用抑制性消减杂交技术构建人甲状腺乳头状癌cDNA消减文库 ,并从中克隆了 3个cDNA片段 ,通过测序 ,同源性分析 ,表明这些片段与来自恶性肿瘤相关基因片段具有同源性 ,提示他们可能在甲状腺癌的发生发展中起到某种重要作用。     

甲状腺激素对仔鼠大脑皮质中NO/cGMP信号转导通路的作用

目的研究甲状腺激素对大鼠仔鼠大脑皮质中一氧化氮(NO)水平、一氧化氮合酶(NOS)活性和环鸟苷酸(cGMP)水平的影响,为探讨甲状腺激素对神经系统发育的作用机制提供实验资料。方法取怀孕3d的SD大鼠10只,随机分为实验组与对照组。实验组饮含1%高氯酸钠的自来水,对照组饮自来水,直至仔鼠产下15d(其产下的仔鼠分别为甲状腺功能低下仔鼠及正常仔鼠)。采用放射免疫法测定仔鼠血清中游离三碘甲状腺原氨酸(FT3)、游离四碘甲状腺原氨酸(FT4)和仔鼠大脑皮质cGMP水平,采用硝酸还原酶法测仔鼠大脑皮质中NO水平,采用分光光度计法测NOS活性。结果实验组仔鼠血清FT3、FT4明显低于对照组(P<0.01)。实验组仔鼠大脑皮质中NO水平、NOS活性及cGMP水平也较对照组显著降低(P<0.01)。相关分析表明:血清FT3与大脑皮质中NO、cGMP水平呈显著正相关,r值分别为0.91、0.97,P<0.01;血清FT4与大脑皮质中NO、cGMP水平也呈显著正相关,r值分别为0.90、0.96,P<0.01;大脑皮质中NO与cGMP水平呈显著正相关,r值为0.90,P<0.01。结论甲状腺激素的缺乏可降低仔鼠大脑皮质中NO、cGMP水平,NO/cGMP信号转导系统在甲状腺功能低下导致脑发育障碍中发挥重要作用。