Leukemia inhibitory factor receptor(LIFR),as a neuroregulatory cytokine receptor,generally shows a neuroprotective effect in central nervous system injuries.In this study,to understand the effect of LIFR on pathogenes...Leukemia inhibitory factor receptor(LIFR),as a neuroregulatory cytokine receptor,generally shows a neuroprotective effect in central nervous system injuries.In this study,to understand the effect of LIFR on pathogenesis of neural tube defects,we explored spatiotemporal expression of LIFR at different stages of fetal development in normal and neural tube defect embryos.Spina bifida aperta was induced with all-trans retinoic acid on embryonic day 10 in rats,and the spatiotemporal expression of LIFR was investigated in spina bifida aperta rats and healthy rats from embryonic day 11 to 17.Real time-polymerase chain reaction and western blot assay were used to examine mRNA and protein expression of LIFR in healthy control and neural tube defect embryos.Results of the animal experiment demonstrated that expression of LIFR protein and mRNA in the spinal cords of normal rat embryos increased with embryonic development.LIFR was significantly downregulated in the spinal cords of spina bifida aperta rats compared with healthy rats from embryonic days 11 to 17.Immunohistochemical staining showed that the expression of LIFR in placenta and spinal cord in spina bifida aperta rat embryos was decreased compared with that in control embryos at embryonic day 15.Results from human embryo specimens showed that LIFR mRNA expression was significantly down-regulated in spinal cords of human fetuses with neural tube defects compared with normal controls at a gestational age of 24 to 33 weeks.The results were consistent with the down-regulation of LIFR in the animal experiments.Our study revealed spatiotemporal changes in expression of LIFR during embryonic neurulation.Thus,LIFR might play a specific role in neural tube development.All animal and human experimental procedures were approved by the Medical Ethics Committee of Shengjing Hospital of China Medical University,China(approval No.2016PS106K)on February 25,2016.展开更多
Environmental and genetic factors influence the occurrence of neural tube defects, such as spina bifida. Specific disease expression patterns will help to elucidate the pathogenesis of disease. However, results obtain...Environmental and genetic factors influence the occurrence of neural tube defects, such as spina bifida. Specific disease expression patterns will help to elucidate the pathogenesis of disease. However, results obtained from animal models, which often exhibit organism specificity, do not fully explain the mechanisms of human spina bifida onset. In the present study, three embryos with a gestational age of approximately 17 weeks and a confirmed diagnosis of spina bifida, as well as 3 age-matched normal embryos, were obtained from abortions. Fetal brain stem tissues were dissected for RNA isolation, and microarray analyses were conducted to examine profiles of gene expression in brain stems of spina bifida and normal embryos using Affymetrix HG-U133A 2.0 GeneChip arrays. Of the 14 500 gene transcripts examined, a total of 182 genes exhibited at least 2.5-fold change in expression, including 140 upregulated and 42 downregulated genes. These genes were placed into 19 main functional categories according to the Gene Ontology Consortium database for biological functions. Of the 182 altered genes, approximately 50% were involved in cellular apoptosis, growth, adhesion, cell cycle, stress, DNA replication and repair, signal transduction, nervous system development, oxidoreduction, immune responses, and regulation of gene transcription. Gene expression in multiple biological pathways was altered in the brain stem of human spina bifida embryos.展开更多
O6-methylguanine DNA methyltransferase(MGMT), a DNA repair enzyme, has been reported in some congenital malformations, but it is less frequently reported in neural tube defects. This study investigated MGMT mRNA expre...O6-methylguanine DNA methyltransferase(MGMT), a DNA repair enzyme, has been reported in some congenital malformations, but it is less frequently reported in neural tube defects. This study investigated MGMT mRNA expression and methylation levels in the early embryo and in different embryonic stages, as well as the relationship between MGMT and neural tube defects. Spina bifida aperta was induced in rats by a single intragastric administration of all-trans retinoic acid on embryonic day(E) 10, whereas normal control rats received the same amount of olive oil on the same embryonic day. DNA damage was assessed by detecting γ-H2 A.X in spina bifida aperta rats. Real time-polymerase chain reaction was used to examine mRNA expression of MGMT in normal control and spina bifida aperta rats. In normal controls, the MGMT mRNA expression decreased with increasing embryonic days, and was remarkably reduced from E11 to E14, reaching a minimum at E18. In the spina bifida aperta model, γ-H2 A.X protein expression was increased, and mRNA expression of MGMT was markedly decreased on E14, E16, and E18. Bisulfite sequencing polymerase chain reaction for MGMT promoter methylation demonstrated that almost all CpG sites in the MGMT promoter remained unmethylated in both spina bifida aperta rats and normal controls, and there was no significant difference in methylation level between the two groups on either E14 or E18. Our results show that DNA damage occurs in spina bifida aperta rats. The mRNA expression of MGMT is downregulated, and this downregulation is independent of promoter DNA methylation.展开更多
Previous studies in our laboratory have demonstrated that the thermosensitivity locus cosegregates with blood pressure and that the elevated expression and restriction fragment length polymorphism of HSP70 gene are as...Previous studies in our laboratory have demonstrated that the thermosensitivity locus cosegregates with blood pressure and that the elevated expression and restriction fragment length polymorphism of HSP70 gene are associ-ated with hypertension. Cell protection against environ-mental stressors such as heat and chemicals is often accom-panied by up-regulated expression of a wide spectrum of heat sliock genes(HSP). To further investigate the interre-lation between HSP expression and blood pressure regulation, we employed an effective method of cloning 2 poten-tial hypertension-related HSPs. Synthetic oligonucleotides corresponding either to a higbly-conserved region of the known HSP family or a repetitive sequence in the protein- encoding gene were used as target primers for polymerase chain reaction (PCR). cDNA prepared from heat-stressed and non-stressed vascular smooth muscle cells (VSMC) of Brown Norway rats (BN.1x) and spontaneously hyperten-sive rats (SHRp) respectively served as template in the reaction. The PCR products were subsequently analyzed in a single-stranded conformational polymorphism (SSCP) electrophoresing system. Differential gene expression in BN.1x and SHRp was seen on autoradiographs of SSCP gel by comparing the migration patterns of PCR-amplified DNA fragments. Using this technique, we also found that HSP27 and a new member of the large HSP gene family were differentially expressed in BN.1x and SHRp VSMC.展开更多
目的·探究小鼠胚胎在视黄酸(retinoic acid,RA)诱导下产生神经管畸形的分子调控机制,揭示小鼠神经管闭合阶段基因表达规律。方法·基于已获得的小鼠胚胎神经管闭合关键期[胚胎发育第8.5日(embryonic day 8.5,E8.5)、E9.5、E10...目的·探究小鼠胚胎在视黄酸(retinoic acid,RA)诱导下产生神经管畸形的分子调控机制,揭示小鼠神经管闭合阶段基因表达规律。方法·基于已获得的小鼠胚胎神经管闭合关键期[胚胎发育第8.5日(embryonic day 8.5,E8.5)、E9.5、E10.5]高质量脑泡转录组数据,利用短时间序列表达挖掘器(Short Time-series Expression Miner,STEM)软件分别得到RA处理组和正常组在3个时间点的基因表达趋势数据。对处理组与正常组基因表达趋势不一致的基因进行基因本体(Gene Ontology,GO)富集分析、京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)富集分析,并随机筛选候选基因以验证测序数据可靠性。利用RA诱导构建神经管畸形小鼠模型,分为处理组和正常组,每组各9只。处理组和正常组孕鼠在E7.5分别接受28 mg/kg RA和香油灌胃处理,在E8.5、E9.5、E10.5收集胎鼠脑泡组织,对筛选的候选基因进行实时荧光定量PCR(quantitative real-time PCR,RT-qPCR)验证。结果·正常组共检测出18255个基因的表达量数据,处理组共检测出19037个基因的表达量数据;正常组基因可归纳至7个具有显著意义的表达模式中,处理组基因可归纳至6个具有显著意义的表达模式中;正常组和处理组检测到表达的基因数目足够、表达的模式相似,具有可比性。进一步分析发现正常组中呈现上升表达趋势但在处理组中呈现下降表达趋势的基因共有46个,在生物学过程层面富集在器官发育、神经元凋亡的正负调控、少突胶质细胞增殖、成纤维生长因子信号通路等;在细胞组分层面,主要参与组成细胞、神经元的基本结构;在分子功能层面,主要与成纤维细胞生长因子受体结合有关。正常组中呈现下降表达趋势而在处理组中呈现上升表达趋势的61个基因,在生物学过程层面富集在细胞溶解、氨基酸/离子转运等功能上;在细胞组分层面,富集在胞内分子、皮质颗粒、胞外区域、细胞间隙等;在分子功能层面,与一系列酶及转运蛋白的活性有关。RT-qPCR验证结果显示转录组测序数据真实可靠。结论·RA干预使小鼠胚胎发育过程中发生基因表达失调和应激反应,导致胚胎发育异常,机体自我保护相关信号通路激活,维持胚胎正常发育的基因受到抑制。展开更多
Background：The most typical cardiac abnormality is conotruncal defects （CTDs） in patients with 22q11 deletion syndrome （22q11DS）.HIRA （histone cell cycle regulator） gene,as one of the candidate genes located at...Background：The most typical cardiac abnormality is conotruncal defects （CTDs） in patients with 22q11 deletion syndrome （22q11DS）.HIRA （histone cell cycle regulator） gene,as one of the candidate genes located at the critical region of 22q11DS,was reported as possibly relevant to CTD in animal models.This study aimed to analyze the level of expression of the HIRA gene in tetralogy of Fallot （TOF） patients and the potential DNA sequence variations in the promoter region.Methods：The messenger RNA （mRNA） expression was examined with quantitative real-time polymerase chain reaction in 39 myocardial tissues of the right ventricular outflow tract （RVOT） from TOF patients and 4 myocardial tissues of RVOT from noncardiac death children.The protein expression was detected using immunohistochemistry in 12 TOF patients and 4 controls.A total of 100 TOF cases and 200 healthy controls were recruited for DNA sequencing.Results：The mRNA and protein expressions of the HIRA gene in the myocardium of the TOF patients were both significantly lower as compared to the controls （P 〈 0.05）.Five single nucleotide polymorphisms （SNPs）,including g.4111A〉G （rs1128399）,g.4265C〉A （rs4585115）,g.4369T〉G （rs2277837）,g.4371C〉A （rs148516780）,and g.4543T〉C （rs111802956）,were found in the promoter region of the HIRA gene.There were no significant differences of frequencies in these SNPs between the TOF patients and the controls （P 〉 0.05）.Conclusion：The abnormal lower expression of the HIRA gene in the myocardium may participate in the pathogenesis of TOF.展开更多
基金supported by the National Natural Science Foundation of China,No.81601292(to DA),No.81671469(to ZWY)the National Basic Research Program of China(973 Program),No.2013CB945402(to ZWY)the National Key Research and Development Program of China,No.2016YFC1000505(to ZWY)
文摘Leukemia inhibitory factor receptor(LIFR),as a neuroregulatory cytokine receptor,generally shows a neuroprotective effect in central nervous system injuries.In this study,to understand the effect of LIFR on pathogenesis of neural tube defects,we explored spatiotemporal expression of LIFR at different stages of fetal development in normal and neural tube defect embryos.Spina bifida aperta was induced with all-trans retinoic acid on embryonic day 10 in rats,and the spatiotemporal expression of LIFR was investigated in spina bifida aperta rats and healthy rats from embryonic day 11 to 17.Real time-polymerase chain reaction and western blot assay were used to examine mRNA and protein expression of LIFR in healthy control and neural tube defect embryos.Results of the animal experiment demonstrated that expression of LIFR protein and mRNA in the spinal cords of normal rat embryos increased with embryonic development.LIFR was significantly downregulated in the spinal cords of spina bifida aperta rats compared with healthy rats from embryonic days 11 to 17.Immunohistochemical staining showed that the expression of LIFR in placenta and spinal cord in spina bifida aperta rat embryos was decreased compared with that in control embryos at embryonic day 15.Results from human embryo specimens showed that LIFR mRNA expression was significantly down-regulated in spinal cords of human fetuses with neural tube defects compared with normal controls at a gestational age of 24 to 33 weeks.The results were consistent with the down-regulation of LIFR in the animal experiments.Our study revealed spatiotemporal changes in expression of LIFR during embryonic neurulation.Thus,LIFR might play a specific role in neural tube development.All animal and human experimental procedures were approved by the Medical Ethics Committee of Shengjing Hospital of China Medical University,China(approval No.2016PS106K)on February 25,2016.
基金Supported by the National Key Project of Scientific and Technical Supporting Programs funded by the Ministry of Science & Technology of China, No. 2007BA107A02the National Basic Research Program of China (973 Program), No. 2007CB511902+2 种基金the Shanxi Scholarship Council of China, No. 2008-48the Shanxi Natural Science Foundation, No. 2010011049-2the National Natural Science Foundation of China, No. 31040056
文摘Environmental and genetic factors influence the occurrence of neural tube defects, such as spina bifida. Specific disease expression patterns will help to elucidate the pathogenesis of disease. However, results obtained from animal models, which often exhibit organism specificity, do not fully explain the mechanisms of human spina bifida onset. In the present study, three embryos with a gestational age of approximately 17 weeks and a confirmed diagnosis of spina bifida, as well as 3 age-matched normal embryos, were obtained from abortions. Fetal brain stem tissues were dissected for RNA isolation, and microarray analyses were conducted to examine profiles of gene expression in brain stems of spina bifida and normal embryos using Affymetrix HG-U133A 2.0 GeneChip arrays. Of the 14 500 gene transcripts examined, a total of 182 genes exhibited at least 2.5-fold change in expression, including 140 upregulated and 42 downregulated genes. These genes were placed into 19 main functional categories according to the Gene Ontology Consortium database for biological functions. Of the 182 altered genes, approximately 50% were involved in cellular apoptosis, growth, adhesion, cell cycle, stress, DNA replication and repair, signal transduction, nervous system development, oxidoreduction, immune responses, and regulation of gene transcription. Gene expression in multiple biological pathways was altered in the brain stem of human spina bifida embryos.
基金supported by the National Natural Science Foundation of China,No.81671469,81171072(to ZWY)the National Basic Research Program of China(973 Program),No.2013CB945402(to ZWY)the Program for Liaoning Innovative Research Team in University of China,No.LT2013016(to ZWY)
文摘O6-methylguanine DNA methyltransferase(MGMT), a DNA repair enzyme, has been reported in some congenital malformations, but it is less frequently reported in neural tube defects. This study investigated MGMT mRNA expression and methylation levels in the early embryo and in different embryonic stages, as well as the relationship between MGMT and neural tube defects. Spina bifida aperta was induced in rats by a single intragastric administration of all-trans retinoic acid on embryonic day(E) 10, whereas normal control rats received the same amount of olive oil on the same embryonic day. DNA damage was assessed by detecting γ-H2 A.X in spina bifida aperta rats. Real time-polymerase chain reaction was used to examine mRNA expression of MGMT in normal control and spina bifida aperta rats. In normal controls, the MGMT mRNA expression decreased with increasing embryonic days, and was remarkably reduced from E11 to E14, reaching a minimum at E18. In the spina bifida aperta model, γ-H2 A.X protein expression was increased, and mRNA expression of MGMT was markedly decreased on E14, E16, and E18. Bisulfite sequencing polymerase chain reaction for MGMT promoter methylation demonstrated that almost all CpG sites in the MGMT promoter remained unmethylated in both spina bifida aperta rats and normal controls, and there was no significant difference in methylation level between the two groups on either E14 or E18. Our results show that DNA damage occurs in spina bifida aperta rats. The mRNA expression of MGMT is downregulated, and this downregulation is independent of promoter DNA methylation.
文摘Previous studies in our laboratory have demonstrated that the thermosensitivity locus cosegregates with blood pressure and that the elevated expression and restriction fragment length polymorphism of HSP70 gene are associ-ated with hypertension. Cell protection against environ-mental stressors such as heat and chemicals is often accom-panied by up-regulated expression of a wide spectrum of heat sliock genes(HSP). To further investigate the interre-lation between HSP expression and blood pressure regulation, we employed an effective method of cloning 2 poten-tial hypertension-related HSPs. Synthetic oligonucleotides corresponding either to a higbly-conserved region of the known HSP family or a repetitive sequence in the protein- encoding gene were used as target primers for polymerase chain reaction (PCR). cDNA prepared from heat-stressed and non-stressed vascular smooth muscle cells (VSMC) of Brown Norway rats (BN.1x) and spontaneously hyperten-sive rats (SHRp) respectively served as template in the reaction. The PCR products were subsequently analyzed in a single-stranded conformational polymorphism (SSCP) electrophoresing system. Differential gene expression in BN.1x and SHRp was seen on autoradiographs of SSCP gel by comparing the migration patterns of PCR-amplified DNA fragments. Using this technique, we also found that HSP27 and a new member of the large HSP gene family were differentially expressed in BN.1x and SHRp VSMC.
基金This work was supported by grants from the Natural Science Foundation of China （No. 81370198, No. 81570283） and National Key Research and Development Program （No. 2016YFC 1000500）.
文摘Background：The most typical cardiac abnormality is conotruncal defects （CTDs） in patients with 22q11 deletion syndrome （22q11DS）.HIRA （histone cell cycle regulator） gene,as one of the candidate genes located at the critical region of 22q11DS,was reported as possibly relevant to CTD in animal models.This study aimed to analyze the level of expression of the HIRA gene in tetralogy of Fallot （TOF） patients and the potential DNA sequence variations in the promoter region.Methods：The messenger RNA （mRNA） expression was examined with quantitative real-time polymerase chain reaction in 39 myocardial tissues of the right ventricular outflow tract （RVOT） from TOF patients and 4 myocardial tissues of RVOT from noncardiac death children.The protein expression was detected using immunohistochemistry in 12 TOF patients and 4 controls.A total of 100 TOF cases and 200 healthy controls were recruited for DNA sequencing.Results：The mRNA and protein expressions of the HIRA gene in the myocardium of the TOF patients were both significantly lower as compared to the controls （P 〈 0.05）.Five single nucleotide polymorphisms （SNPs）,including g.4111A〉G （rs1128399）,g.4265C〉A （rs4585115）,g.4369T〉G （rs2277837）,g.4371C〉A （rs148516780）,and g.4543T〉C （rs111802956）,were found in the promoter region of the HIRA gene.There were no significant differences of frequencies in these SNPs between the TOF patients and the controls （P 〉 0.05）.Conclusion：The abnormal lower expression of the HIRA gene in the myocardium may participate in the pathogenesis of TOF.
