帕金森病人血清分化型胚胎软骨表达基因1和肝X受体β水平的相关性研究

目的:探讨帕金森病(PD)病人血清中分化型胚胎软骨表达基因1(DEC1)和肝X受体β(LXRβ)水平及二者相关性。方法:选择PD病人50例(PD组),选择同期健康人群50名为对照(对照组)。检测2组受试者血清DEC1、LXRβ水平。结果:PD组血清DEC1水平明显高于对照组(P<0.01),LXRβ水平明显低于对照组(P<0.01)。不同性别的PD病人DEC1和LXRβ水平差异均无统计学意义(P>0.05);年龄≥60岁PD病人的LXRβ水平低于<60岁者(P<0.05);有认知功能障碍的PD病人DEC1和LXRβ水平分别明显高于和低于无认知障碍者(P<0.01);随病情分期加重,PD病人的DEC1水平逐渐升高,LXRβ水平逐渐降低(P<0.05~P<0.01)。PD病人血清DEC1水平与LXRβ水平呈负相关关系(r=-0.326,P<0.05)。结论:PD病人血清DEC1与LXRβ水平呈负相关关系,二者与PD的发病及病情进展有关。

致密化相关基因在动物胚胎发育过程中的作用研究进展

桑椹胚致密化是早期胚胎发育过程中的关键步骤,与胚胎质量密切相关。已有研究发现体内外发育的胚胎表现明显不同的致密化过程,缝隙连接蛋白、上皮钙调素蛋白基因的表达也有差异。本文针对致密化相关基因在胚胎早期发育过程中的作用及一些影响因素的研究进展进行综述。

绵羊发育过程的遗传调控

目前已测出9个oTP-1的cDNA序列,这些序列含有功能不同的基因,这些基因的共有序列含有一个595bp的ORF,编码23个氨基酸信号肽,接着是一个172个氨基酸的成熟蛋白。这些基因在妊娠早期分别转录,对不同的子宫刺激因子有所反应。绵羊胚胎基因组于8～16细胞阶段激活。在此之前,胚胎依赖于卵母细胞内的母体基因,从母体控制转换为胚胎控制,要经历3个关键步骤。

Activation of paternally expressed imprinted genes in newly derived germline-competent mouse parthenogenetic embryonic stem cell lines

Parthenogenetic embryonic stem （pES） cells provide a valuable in vitro model system for studying the molecular mechanisms that underlie genomic imprinting. However, the pluripotency of pES cells and the expression profiles of paternally expressed imprinted genes have not been fully explored. In this study, three mouse pES cell lines were established and the differentiation potential of these cells in extended culture was evaluated. The undifferentiated cells had a normal karyotype and homozygous genome, and expressed ES-cell-specific molecular markers. The cells remained undifferentiated after more than 50 passages and exhibited pluripotent differentiation capacity. All three lines of the established ES cells produced teratomas; two lines of ES cells produced chimeras and germline transmission. Furthermore, activation of the paternally expressed imprinted genes Snrpn, U2afl-rsl, Peg3, Impact, Zfp127, Dlkl and Mest in these cells was detected. Some paternally expressed imprinted genes were found to be expressed in the blastocyst stage of parthenogenetically activated embryos in vitro and their expression level increased with extended pES cell culture. Furthermore, our data show that the activation of these paternally expressed imprinted genes in pES cells was associated with a change in the methylation of the related differentially methylated regions. These findings provide direct evidence for the pluripotency of pES cells and demonstrate the association between the DNA methylation pattern and the activa- tion of paternally expressed imprinted genes in pES cells. Thus, the established ES cell lines provide a valuable model for studying epigenetic regulation in mammalian development.

Developmental expression of Cyclin H and Cdk7 in zebrafish: the essential role of Cyclin H during early embryo development

Cyclin-dependent kinase 7 （Cdk7） is the catalytic subunit of the metazoan Cdk-activating kinase （CAK）. Activation of Cdk7 requires its association with a regulatory subunit, Cyclin H. Although the Cdk7/Cyclin H complex has been implicated in the regulation ofRNA polymerase in several species, the precise function of their orthologs in zebrafish has not been fully elucidated. In this study, we isolated from zebrafish blastula embryos two cDNAs encoding the orthologs of human Cyclin H and Cdk7, and examined the role of Cdk7/Cyclin H in zebrafish embryogenesis. Sequence analysis showed that the zebrafish Cyclin H and Cdk7 cDNAs encode proteins with 65% and 86% identity to the respective hu- man orthologs. RT-PCR and whole-mount in situ hybridization analyses of their expression in unfertilized eggs, embryos and organs of adult fish suggested that Cyclin H and Cdk7 messages are maternally loaded. Our data also showed that their transcripts were detected throughout development. Distribution of Cyclin H transcripts was found to be ubiquitous during early stages of development and become restricted to the anterior neural tube, brain, eyes, procreate tissues, liver and heart by 5 days post-fertilization. Expression of a dominant-negative form of Cyclin H delayed the onset of zygotic transcription in the early embryo, resulting in apoptosis at 5 hours post-fertilization and leading to sever defects in tis- sues normally exhibiting high levels of Cyclin H expression. These results implicate Cyclin H in the regulation of the transcriptional machinery during midblastula transition and suggest that it is an essential gene in early zebrafish larval development.

The Study on the Gene Expression of Preimplantation IVF Bovine Embryos

The cattle different stage embryos obtained from in vitro was studied using the technology of single preimplantation embryo mRNA different display:single 8-cell and blastocyst stage embryos were studied using technology of mRNA different display and one different fragment was found. The result suggested that this fragment displayed high homology (99%) to cattle mRNA for ribosomal protein L31. Then to detect the expression of RPL31mRNA in 8 cell and blastocyst stage embryos by real-time quantitative PCR,the result showed the relative amount of 8 cells was 3.2 times of blastocyst's.

Regulated expression of TATA-binding protein-related factor 3(TRF3)during early embryogenesis

RNA polymerase （Pol） Ⅱ transcription persists in TATA-box-binding protein （TBP）^-/- mutant mouse embryos, indicating TBP-independent mechanisms for Pol Ⅱ transcription in early development. TBP-related factor 3 （TRF3） has been proposed to substitute for TBP in TBP^-/- mouse embryos. We examined the expression of TRF3 in maturing oocytes and early embryos and found that TRF3 was co-expressed with TBP in the meiotic oocytes and early embryos from the late one-cell stage onward. The amounts of TBP and TRF3 changed dynamically and correlated well with transcriptional activity. Chromatin immunoprecipitation （CHIP） assay revealed that different gene promoters in mouse embryonic stem （ES） cells recruited TRF3 and TBP selectively. Comparative analyses of TRF3 and TBP during cell cycle showed that both factors proceeded through cell cycle in a similar pace, except that TRF3 was slightly delayed than TBP in entering the nucleus when cells were exiting the M-phase. Data from expression and biochemical analyses therefore support the hypothesis that TRF3 plays a role in early mouse development. In addition, results from co-localization study suggest that TRF3 may be also involved in Pol Ⅰ transcription.

Amphibian metamorphosis as a model for studying the developmental actions of thyroid hormone

The thyroid hormones L-thyroxine and triiodo-L-thyronine have profound effects on postembryonic development of most vertebrates. Analysis of their action in mammals is vitiated by the exposure of the developing foetus to a number of maternal factors which do not allow one to specifically define the role of thyroid hormone (TH)or that of other hormones and factors that modulate its action. Amphibian metamorphosis is obligatorily dependent on TH which can initiate all the diverse physiological manifestations of this postembryonic developmental process(morphogenesis, cell death, re-structuring, etc.) in free-living embryos and larvae of most anurans. This article will first describe the salient features of metamorphosis and its control by TH and other hormones. Emphasis will be laid on the key role played by TH receptor (TR), in particular the phenomenon of TR gene autoinduction, in initiating the developmental action of TH. Finally, it will be argued that the findings on the control of amphibian metamorphosis enhance our understanding of the regulation of postembryonic development by TH in other vertebrate species.

Cloning and expression patterns of two Smad genes during embryonic development and shell formation of the Pacific oyster Crassostrea gigas

Increasing evidence indicates that transforming growth factor β （TGF-β） signaling pathways play many important roles in the early development of mollusks. However, limited information is known concerning their detailed mechanisms. Here, we describe the identification, cloning and characterization of two Smad genes, the key components of TGF-β signaling pathways, from the Pacific oyster Crassostrea gigas. Sequence analysis of the two genes, designated as cgi-smad1/5/8 and cgi-smad4, revealed conserved functional characteristics. The two genes were widely expressed in embryos and larvae, suggesting multiple roles in the early development of C. gigas. The mRNA of the two genes aggregated in the D quadrant and cgi-smad4 was highly expressed on the dorsal side of the gastrula, indicating that TGF-β signaling pathways may be involved in dorsoventral patterning in C. gigas. Furthermore, high expression levels of the two genes in the shell fields of embryos at different stages suggested important roles for TGF-β signaling pathways in particular phases of shell development, including the formation of the initial shell field and the biomineralization of larval shells. The results of this study provide fundamental support for elucidating how TGF-β signaling pathways participate in the early development of bivalve mollusks, and suggest that further work is warranted to this end.

Differentiation of mouse embryonic stem cells into insulin-secreting cells in vitro

Regenerative medicine, including cell-replacement strategies, may have an important role in the treatment of type 1 diabetes which is associated with decreased islet cell mass. To date, significant progress has been made in generating insulin-secreting 13 cells from pluripotent mouse embryonic stem cells （ESCs）.The aim of this study is to explore the potential of regulating the differentiation of ESCs into pancreatic endocrine cells capable of synthesizing the pancreatic hormones including insulin, glucagon, somatostatin and pancreatic polypeptide under proper conditions. Undifferentiated ES cell line was stably transfected with mouse RIP-YFP plasmid construction in serum-free medium using LipofectamineTM 2000 Reagents. We tested pancreatic specific gene expression and characterized these ESC-derived pancreatic endocrine cells. Most of these insulin-secreting cells co-expressed many of the phenotypic markers characteristic of 13 cells such as insulinl, insulin2, Isletl, MafA, insulinoma-associated antigen 1 （IA1） and so on, indicating a similar gene expression pattern to adult islet 13 cells in vivo. Characterization of this population revealed that it consisted predominantly of pancreatic endocrine cells that were able to undergo pancreatic specification under the appropriate conditions. We also demonstrated that zinc supplementation mediated up-regulation of insulin-secreting cells as an effective inducer promoted the development of ESC-derived diabetes therapy. In conclusion, this work not only established an efficient pancreatic differentiation strategy from ESCs to pancreatic endocrine lineage in vitro, but also leaded to the development of new strategies to derive transplantable islet-replacement 13 cells from embryonic stem cells for the future applications of a stem cell based therapy of diabetes.

Gankyrin expression during mouse embryogenesis

Objective: To observe the gene expression of Gankyrin during mouse embryogenesis and reveal the gene biological significance during organs and tissues formation. Methods: The expressions of Gankyrin mRNA in various organs and tissues were detected by in situ hybridization at indicated times during embryogenesis. Results: The expression of Gankyrin mRNA in mouse day 12.5 embryo was mainly in midbrain, interbrain and endbrain; in mouse day 14.5 embryo mainly in midbrain, aorta, liver, gonad, cranium and rib; in mouse day 16.5 embryo mainly in cranium, rib and vertebra; and in mouse day 18.5 embryo mainly in cranium, rib and intestinal mucosa. Conclusion: Gankyrin gene probably participates in the development of the neural tissues (such as midbrain, interbrain and endbrain etc.), aorta, liver and gonad, intestinal mucosa and bone tissues, which may be closely associated with the function of the organs and tissues.

Identification of a NF-кB site in the negative regulatory element (ε-NRAII) of human ε-globin gene and its binding protein NF-кB p50 in the nuclei of K562 cells

The developmental control of the human e-globin gene expression is mediated by transcription regulatory elements in the 5’ flanking DNA of this gene. Sequence analysis has revealed a DNA motif (GGGGAATTTGCT) similar to NF-кB consensus sequence resides in the negative regulatory element (-3028bp～ -2902bp, termed ε-NRAII) 5’ to the cap site of this gene. NRF DNA fragment (-3010bp～ -2986bp) containing the NF-кB motif similar sequence was synthesized and used in electrophoresis mobility shift assay (EMSA) and competitive analysis. Data showed that a protein factor from nuclear extracts of K562 cells specifically interacted with NRF DNA fragment. The synthetic NF DNA fragment (containing NF-кB consensus sequence) could competed for the protein binding, but MNF DNA fragment (mutated NF-кB motif) could not, suggesting that the binding protein is a member of NF-кB/Rel family. Western blot assay demonstrated that the molecular weight of NF-кB protein in the nuclei of K562 cells is 50ku. We suggested that NF-кB p50 may play an important role in the regulation of human c-globin gene expression.

An Arabidopsis embryonic lethal mutant with reducedexpression of alanyl-tRNA synthetase gene

In present paper, one of the T-DNA insertional embryonic lethal mutant of Arabidopsis is identifled and designated as acd mutant. The embryo development of this mutant is arrested in globular stage. The cell division pattern is abnormal during early embryogenesis and results in disturbed cellular differentiation. Most of mutant embryos are finally degenerated and aborted in globular stage. However, a few of them still can germinate in agar plate and produce seedlings with shorter hypocotyl and distorted shoot meristem. To understand the molecular basis of the phenotype of this mutant, the joint fragment of T-DNA/plant DNA is isolated by plasmid rescue and Dig-labeled as probe for cDNA library screening. According to the sequence analysis and similarity searching, a 936bp cDNA sequence (EMBL accession#: Y12555) from selected positive clone shows a 99.8 % (923/925bp) sequence homology with Alanyl-tRNA Synthetase (A1aRS) gene of Arabidopsis thaliana. Furthermore, the data of in sitll hybridization experiment indicate that the expression of AlaRS gene is weak in early embryogenesis and declines along with globular embryo ’development’ in this mutant.Accordingly, the reduced expression of AlaRS gene maybe closely related to the morphological changes in early embryogenesis of this lethal mutant.

EGFR与DEC1蛋白共表达促进肺腺癌肿瘤细胞淋巴结转移

目的探讨EGFR和DEC1在肺腺癌中的表达及与临床特征的关系。方法采用免疫组织化学方法检测75例肺腺癌癌组织及癌旁组织EGFR和DEC1的表达。结果在癌组织中,EGFR胞膜上的表达为45.3%(34/75),DEC1胞核上的表达为54.7%(41/75),两者均与癌旁组织的表达有统计学差异(P<0.001);EGFR的表达与组织分化(P=0.033)、淋巴结转移(P=0.006)和远处转移(P=0.043)相关,DEC1与年龄(P=0.026)和肿瘤大小(P=0.047)相关。表达模式为EGFR+/DEC1+的肺腺癌患者比EGFR-/DEC1-的患者发生淋巴结转移的百分率高,两者有统计学差异(P=0.024)。结论 EGFR和DEC1蛋白的共表达可促进肺腺癌患者淋巴结转移。

m^(6)A reader Igf2bp3 enables germ plasm assembly by mA-dependent regulation of gene expression in zebrafish

Bucky ball(Buc)is involved in germ plasm(GP)assembly during early zebrafish development by regulating GP mRNA expression via an unknown mechanism.The present study demonstrates that an m^(6)A reader Igf2bp3 interacts and colocalizes with Buc in the GP.Similar to the loss of Buc,the genetic deletion of maternal igf2bp3 in zebrafish leads to abnormal GP assembly and insufficient germ cell specification,which can be partially restored by the injection of igf2 bp3 mRNA.Igf2bp3 binds to m^(6)A-modified GPorganizer and GP mRNAs in an m^(6)A-dependent manner and prevents their degradation.These findings indicate that the functions of Igf2bp3,a direct effector protein of Buc,in GP mRNA expression and GP assembly involve m^(6)A-dependent regulation;these results emphasize a critical role of m^(6)A modification in the process of GP assembly.

Maternal Eomesodermin regulates zygotic nodal gene expression for mesendoderm induction in zebrafish embryos

Development of animal embryos before zygotic genome activation at the mid blastula transition （MBT） is essentially supported by eggderived maternal products. Nodal proteins are crucial signals for mesoderm and endoderm induction after the MBT. It remains unclear which maternal factors activate zygotic expression of nodal genes in the ventrotateral blastodermal margin of the zebrafish blastulas. In this study, we show that loss of maternal Eomesodermin a （Eomesa）, a T-box transcription factor, impairs zygotic expression of the nodal genes ndr1 and ndr2 as well as mesodermal and endodermal markers, indicating an involvement in mesendoderm induction. Maternal Eomesa is also required for timely zygotic expression of the transcription factor gene mxtx2, a regulator of nodal gene expression. Eomesa directly binds to the Eomes-binding sites in the promoter or enhancer of ndr1, ndr2, and rnxtx2 to activate their transcrip- tion. Furthermore, human and mouse Nodal genes are also regulated by Eomes. Transfection of zebrafish eomesa into murine embryonic stem cells promotes mesendodermal differentiation with constant higher levels of endogenous Nodal expression, suggesting a conserved function of Eomes. Taken together, our findings reveal a conserved rote of maternal T-box transcription factors in regulating nodal gene expression and mesendoderm induction in vertebrate embryos.

Growth of embryo and gene expression of nutrient transporters in the small intestine of the domestic pigeon(Columba livia)

The objective of this study was to investigate the relationship between gene expression of nutrient（amino acid, peptide, sodium and proton） transporters in the small intestine and embryonic growth in domestic pigeons（Columba livia）. One hundred and twenty-five fertilized eggs were randomly assigned into five groups and were incubated under optimal conditions（temperature of 38.1 °C and relative humidity of 55%）. Twenty embryos/birds from each group were sacrificed by cervical dislocation on embryonic day（E） 9, 11, 13, 15 and day of hatch（DOH）. The eggs, embryos（without yolk sac）, and organs（head, brain, heart, liver, lungs, kidney, gizzard, small intestine, legs, and thorax） were dissected, cleaned, and weighed. Small intestine samples were collected for RNA isolation. The m RNA abundance of intestinal nutrient transporters was evaluated by real-time reverse transcription-polymerase chain reaction（RT-PCR）. We classified these ten organs into four types according to the changes in relative weight during embryonic development. In addition, the gene expression of nutrient transporters was differentially regulated by embryonic day. The m RNA abundances of b^0,＋AT, EAAT3, y^＋LAT2, Pep T1, LAT4, NHE2, and NHE3 increased linearly with age, whereas m RNA abundances of CAT1, CAT2, LAT1, EAAT2, SNAT1, and SNAT2 were increased to higher levels on E9 or E11 and then decreased to lower levels until DOH. The results of correlation analysis showed that the gene expressions of b^0,＋AT, EAAT3, Pep T1, LAT4, NHE2, NHE3, and y^＋LAT2 had positive correlations with body weight（0.71〈correlation coefficient（CC）〈0.82, P〈0.0001）, while CAT1, CAT2, EAAT2, SNAT1, and SNAT2 had negative correlations with body weight（-0.86〈CC〈-0.64, P〈0.0001）. The gene expressions of b^0,＋AT, EAAT3, LAT4, Pep T1, NHE2, NHE3, and y^＋LAT2 showed positive correlations with intestinal weight（0.80〈CC〈0.91, P〈0.0001）, while CAT1, CAT2, and EAAT2 showed negative correlations with intestinal weight（-0.84〈CC〈-0.67, P〈0.0001）. It was concluded that the differences between growth trajectories of organs and gene expression of nutrient transporters in small intestine were due to their functional and physiological properties, which provided a comprehensive study of amino acid and peptide transporter m RNA in the small intestine during embryonic growth of pigeons.

Acidic pH transiently prevents the silencing of self-renewal and dampens microRNA function in embryonic stem cells

Enhanced glycolysis is a distinct feature associated with numerous stem cells and cancer cells.However,little is known about its regulatory roles in gene expression and cell fate determination.Here,we confirm that glycolytic metabolism and lactate production decrease during the differentiation of mouse embryonic stem cells(mESCs).Importantly,acidic pH due to lactate accumulation can transiently prevent the silencing of mESC self-renewal in differentiation conditions.Furthermore,acidic pH partially blocks the differentiation of human ESCs(hESCs).Mechanistically,acidic pH downregulates AGO1 protein and de-represses a subset of mRNA targets of miR-290/302 family of microRNAs which facilitate the exit of naive pluripotency state in mESCs.Interestingly,AGO1 protein is also downregulated by acidic pH in cancer cells.Altogether,this study provides insights into the potential function and underlying mechanism of acidic pH in pluripotent stem cells(PSCs).