为进一步丰富鱼类MHC class Ⅱ基因的研究,同时也为进一步探讨低磷饲料中添加维生素D3对鱼类免疫功能可能的影响,实验利用RACE(Rapid-amplification of c DNA ends)即c DNA末端快速扩增技术,成功克隆出黄颡鱼(Pelteobagrus fulvidraco)...为进一步丰富鱼类MHC class Ⅱ基因的研究,同时也为进一步探讨低磷饲料中添加维生素D3对鱼类免疫功能可能的影响,实验利用RACE(Rapid-amplification of c DNA ends)即c DNA末端快速扩增技术,成功克隆出黄颡鱼(Pelteobagrus fulvidraco)主要组织相容性复合体(Major histocompatibility complex,MHC)class Ⅱ抗原基因,全长1074 bp,其中ORF(Open reading frame)708 bp,编码236个氨基酸,5′UTR(5′端非翻译区)78 bp,3′UTR(3′端非翻译区)259 bp。进行氨基酸序列比对分析得到:黄颡鱼MHC class Ⅱ基因ORF氨基酸序列与长吻逘(Leiocassis longirostris)的氨基酸序列相似度最高为69.5%,与锦鲤(Cyprinus carpio)的氨基酸序列相似度最低为50.4%。利用q PCR对黄颡鱼MHC class Ⅱ基因进行组织表达分析,结果表明MHC class Ⅱ在小肠、肝脏、鳃中表达较高;在肌肉、鳍条中表达较低;而在肾、脾脏、脑、头肾中表达量极低(几乎检测不到)。在低磷饲料中添加维生素D3显著诱导了该基因的上调表达。研究结果展示了黄颡鱼MHC class Ⅱ抗原基因的分子结构、组织表达以及维生素D3的作用,在降低磷排放的同时,为今后黄颡鱼免疫抗病及分子选育等方向的深入研究及免疫型饲料的使用奠定了基础。展开更多
AIM： To investigate the role of MHC class Ⅱ in the modulation of gastric epithelial cell apoptosis induced by H pylori infection. METHODS： After stimulating a human gastric epithelial cell line with bacteria or ago...AIM： To investigate the role of MHC class Ⅱ in the modulation of gastric epithelial cell apoptosis induced by H pylori infection. METHODS： After stimulating a human gastric epithelial cell line with bacteria or agonist antibodies specific for MHC class Ⅱ and CD95, the quantitation of apoptotic and anti-apoptotic events, including caspase activation, BCL-2 activation, and FADD recruitment, was performed with a fluorometric assay, a cytometric bead array, and confocal microscopy, respectively. RESULTS： Pretreatment of N87 cells with the anti-MHC class ⅡIgM antibody RFD1 resulted in a reduction in global caspase activation at 24 h of H pylori infection. When caspase 3 activation was specifically measured, crosslinking of MHC class Ⅱ resulted in markedly reduced caspase activation, while simple ligation of MHC class Ⅱ did not. Crosslinking of MHC class Ⅱ also resulted in an increased activation of the anti-apoptosis molecule BCL-2 compared to simple ligation. Confocal microscope analysis demonstrated that the pretreatment of gastric epithelial cells with a crosslinking anti-MHC class Ⅱ IgM blocked the recruitment of FADD to the cell surface. CONCLUSION： The ability of MHC class Ⅱ to modulate gastric epithelial apoptosis is at least partially dependent on its crosslinking. The crosslinking of this molecule has anti-apoptotic effects during the earlier time points of Hpylori infection. This effect is possibly mediated by the ability of MHC class Ⅱ to modulate the activation of the pro-apoptotic receptor Fas by blocking the recruitment of the accessory molecule FADD, and this delay in apoptosis induction could allow for prolonged cytokine secretion by Hpylori-infected gastric epithelial cells.展开更多
AIM： TO investigate the role of MHC class Ⅱ in the modulation of gastric epithelial cell apoptosis induced by H pylori infection. METHODS： After stimulating a human gastric epithelial cell line with bacteria or ago...AIM： TO investigate the role of MHC class Ⅱ in the modulation of gastric epithelial cell apoptosis induced by H pylori infection. METHODS： After stimulating a human gastric epithelial cell line with bacteria or agonist antibodies specific for MHC class Ⅱ and CD95, the quantitation of apoptotic and anti-apoptotic events, including caspase activation, BCL-2 activation, and FADD recruitment, was performed with a fluorometric assay, a cytometric bead array, and confocal microscopy, respectively. RESULTS： Pretreatment of N87 cells with the anti-MHC class Ⅱ IgM antibody RFD1 resulted in a reduction in global caspase activation at 24 h of H pylori infection. When caspase 3 activation was specifically measured, crosslinking of MHC class Ⅱ resulted in a marked reduced caspase activation, while simple ligation of HHC class Ⅱ did not. Crosslinking of HHC class Ⅱ also resulted in an increased activation of the anti-apoptosis molecule BCL-2 compared to simple ligation. Confocal microscope analysis demonstrated that the pretreatment of gastric epithelial cells with a crosslinking anti-HHC class Ⅱ IgH blocked the recruitment of FADD to the cell surface. CONCLUSION： The results presented here demonstrate that the ability of MHC class Ⅱ to modulate gastric epithelial apoptosis is at least partially dependent on its crosslinking. Furthermore, while previous research has demonstrated that MHC class Ⅱ signaling can be proapoptotic during extended ligation, we have shown that the crosslinking of this molecule has anti-apoptotic ef-fects during the earlier time points of Hpylori infection. This effect is possibly mediated by the ability of MHC class Ⅱ to modulate the activation of the pro-apoptotic receptor Fas by blocking the recruitment of the accessory molecule FADD, and this delay in apoptosis induction could allow for prolonged cytokine secretion by Hpyloriinfected gastric epithelial cells.展开更多
Purpose:To investigate the density, distribution and morphology of macrophages and MHC class II -positive dendritic cells in the iris and ciliary body of lewis rats. Methods:Immunohistochemistry was performed using mo...Purpose:To investigate the density, distribution and morphology of macrophages and MHC class II -positive dendritic cells in the iris and ciliary body of lewis rats. Methods:Immunohistochemistry was performed using monoclonal antibodies specific to monocytes and macrophages (ED1,ED2) and MHC class II -positive cells (OX6) on wholemounts of the iris-ciliary body complex isolated form normal lewis rats.Results:A well developed network of macrophages was present in the iris and ciliary body of normal lewis rats. These cells, morphologically displaying dendriti-form or pleiomorphic appearance, were more densely arranged in mid-iris (950 + 189 cells/mm2) than in iris base (482 ± 78 cells/mm2) and pupil margin (595 ± 92 cells/mm2). A similar network of MHC class II -positive cells with a cell density 452 ± 78 cells/mm2 was almost uniformly distributed in the iris of normal lewis rats.Conclusions : A network of macrophages and MHC class II -positive cells was established in the iris and ciliary body of展开更多
A fragment spanning over exon 2 and intron 2 of major histocompatibility complex B-LB Ⅱ genes was amplified using PCR, cloned and sequenced in 13 individuals from eight Chinese indigenous chicken breeds and one intro...A fragment spanning over exon 2 and intron 2 of major histocompatibility complex B-LB Ⅱ genes was amplified using PCR, cloned and sequenced in 13 individuals from eight Chinese indigenous chicken breeds and one introduced breed. Another 41 sequences of MHC class Ⅱ β from ten vertebrate species were cited from the NCBI GenBank. Thirteen new B-LB Ⅱ alleles were found in the chicken breeds sampled. Alignment of the exon 2 sequences revealed 91.1-97.8% similarity to each other within the chickens sampled, and the chickens shared 84.1-87.0% homology to Phasianus colchicus, 78.5-81.5% similarity to Coturnix japonica. The sequences in poultry showed 62.6-68.1% identity to HLA-DRB1, 50-61.5% similarity to DQB (HLA-, SLA- and H2-BB), 53.7-60% to HLA-DPB and 53.3-57.8% similarity to HLA-DOB. The frequency of nonsynonymous substitutions of nucleotide was higher than that of synonymous substitutions, and the frequencies of nonsynonymous and synonymous substitutions in poultry B-LB Ⅱ genes were lower than those observed in mammalian DRB1 and DQB1 genes. The deduced amino acid sequences of MHC class Ⅱ β1 domain exhibited extreme difference in conversed region and variable region patterns among the various species, but the two conserved cysteines forming disulfide-bond were shown consistent in poultry with that in mammalian species; and the carbohydrate attachment site was found more conserved in chicken, Homo sapiens, Bos taurus, Ovis aries and Capra hircus than in Sus scrofa and rodent animals. Compared with exon 2 of DQB1 genes of Homo sapiens, ruminant species and Sus scrofa, the differentia that the deletion of six nucleotides at position195 to 200 of exon 2 of DQB1 genes, and insertion of three nucleotides at position 247 to 249 of the exon 2 existed in rodent species were found, which led to the absence of three AA residues at position 65, 66, and 67 within β1 domain of DQB1 chain, and the insertion of one AA residue at position 85. The difference of the deletion of six nucleotides at position 72 to 77 of exon 2 of DPB1 genes was observed with Homo sapiens DQB1, which caused absence of three AA residues at position 24, 25, and 26 of β1 domain of DPB1 chain. The phylogenetic tree revealed that the B-LB Ⅱ sequences from poultry are not orthologous to the class Ⅱ MHC β-chain genes of mammalian species. The tree indicated that genetic evolutionary relationship of chickens with Phasianus colchicus was much closer than with Coturnix japonica, and the DQB and DPB clusters are more tightly related to each other than to the remaining clusters.展开更多
The worldwide declines in amphibian populations have largely been caused by infectious fungi and bacteria. Given that vertebrate immunity against these extracellular pathogens is primarily functioned by the major hist...The worldwide declines in amphibian populations have largely been caused by infectious fungi and bacteria. Given that vertebrate immunity against these extracellular pathogens is primarily functioned by the major histocompatibility complex（MHC） class Ⅱ molecules, the characterization and the evolution of amphibian MHC class Ⅱ genes have attracted increasing attention. The polymorphism of MHC class Ⅱ genes was found to be correlated with susceptibility to fungal pathogens in many amphibian species, suggesting the importance of studies on MHC class Ⅱ genes for amphibians. However, such studies on MHC class Ⅱ gene evolution have rarely been conducted on amphibians in China. In this study, we chose Omei treefrog（Rhacophorus omeimontis）, which lived moist environments easy for breeding bacteria, to study the polymorphism of its MHC class Ⅱ genes and the underlying evolutionary mechanisms. We amplified the entire MHC class ⅡB exon 2 sequence in the R. omeimontis using newly designed primers. We detected 102 putative alleles in 146 individuals. The number of alleles per individual ranged from one to seven, indicating that there are at least four loci containing MHC class ⅡB genes in R. omeimontis. The allelic polymorphism estimated from the 102 alleles in R. omeimontis was not high compared to that estimated in other anuran species. No significant gene recombination was detected in the 102 MHC class ⅡB exon 2 sequences. In contrast, both gene duplication and balancing selection greatly contributed to the variability in MHC class ⅡB exon 2 sequences of R. omeimontis. This study lays the groundwork for the future researches to comprehensively analyze the evolution of amphibian MHC genes and to assess the role of MHC gene polymorphisms in resistance against extracellular pathogens for amphibians in China.展开更多
为了阐明草鱼MHC class I等位基因的结构与多态性,进一步研究其与疾病的关系,从草鱼cDNA文库中克隆了MHC class I基因(Ctid-MHC I);并通过对12个个体Ctid-MHC I的克隆,分析了其等位基因的多态性与三级结构.结果显示Ctid-MHC I等位基因在...为了阐明草鱼MHC class I等位基因的结构与多态性,进一步研究其与疾病的关系,从草鱼cDNA文库中克隆了MHC class I基因(Ctid-MHC I);并通过对12个个体Ctid-MHC I的克隆,分析了其等位基因的多态性与三级结构.结果显示Ctid-MHC I等位基因在α1与α2区域变异幅度大,可分为6类(Ctid-MHC I-UA-UF),9型(A-I);但是其三级结构和抗原多肽结合的关键性氨基酸十分保守.结果阐明了草鱼MHC class I分子在8个区域(A-H)置换率高,存在插入或缺失以及长度变异,使等位基因呈现高度多态性.动物MHC class I分子系统树也提示了我国大陆架上鱼类、两栖类、鸟类、哺乳动物和人的遗传距离与分枝年代.展开更多
文摘为进一步丰富鱼类MHC class Ⅱ基因的研究,同时也为进一步探讨低磷饲料中添加维生素D3对鱼类免疫功能可能的影响,实验利用RACE(Rapid-amplification of c DNA ends)即c DNA末端快速扩增技术,成功克隆出黄颡鱼(Pelteobagrus fulvidraco)主要组织相容性复合体(Major histocompatibility complex,MHC)class Ⅱ抗原基因,全长1074 bp,其中ORF(Open reading frame)708 bp,编码236个氨基酸,5′UTR(5′端非翻译区)78 bp,3′UTR(3′端非翻译区)259 bp。进行氨基酸序列比对分析得到:黄颡鱼MHC class Ⅱ基因ORF氨基酸序列与长吻逘(Leiocassis longirostris)的氨基酸序列相似度最高为69.5%,与锦鲤(Cyprinus carpio)的氨基酸序列相似度最低为50.4%。利用q PCR对黄颡鱼MHC class Ⅱ基因进行组织表达分析,结果表明MHC class Ⅱ在小肠、肝脏、鳃中表达较高;在肌肉、鳍条中表达较低;而在肾、脾脏、脑、头肾中表达量极低(几乎检测不到)。在低磷饲料中添加维生素D3显著诱导了该基因的上调表达。研究结果展示了黄颡鱼MHC class Ⅱ抗原基因的分子结构、组织表达以及维生素D3的作用,在降低磷排放的同时,为今后黄颡鱼免疫抗病及分子选育等方向的深入研究及免疫型饲料的使用奠定了基础。
基金the National Institutes of Health Grants DK50669 and DK56338. EB was a recipient of a fellowship under National Institutes of Health T32 AI007536-06 Training Grant
文摘AIM： To investigate the role of MHC class Ⅱ in the modulation of gastric epithelial cell apoptosis induced by H pylori infection. METHODS： After stimulating a human gastric epithelial cell line with bacteria or agonist antibodies specific for MHC class Ⅱ and CD95, the quantitation of apoptotic and anti-apoptotic events, including caspase activation, BCL-2 activation, and FADD recruitment, was performed with a fluorometric assay, a cytometric bead array, and confocal microscopy, respectively. RESULTS： Pretreatment of N87 cells with the anti-MHC class ⅡIgM antibody RFD1 resulted in a reduction in global caspase activation at 24 h of H pylori infection. When caspase 3 activation was specifically measured, crosslinking of MHC class Ⅱ resulted in markedly reduced caspase activation, while simple ligation of MHC class Ⅱ did not. Crosslinking of MHC class Ⅱ also resulted in an increased activation of the anti-apoptosis molecule BCL-2 compared to simple ligation. Confocal microscope analysis demonstrated that the pretreatment of gastric epithelial cells with a crosslinking anti-MHC class Ⅱ IgM blocked the recruitment of FADD to the cell surface. CONCLUSION： The ability of MHC class Ⅱ to modulate gastric epithelial apoptosis is at least partially dependent on its crosslinking. The crosslinking of this molecule has anti-apoptotic effects during the earlier time points of Hpylori infection. This effect is possibly mediated by the ability of MHC class Ⅱ to modulate the activation of the pro-apoptotic receptor Fas by blocking the recruitment of the accessory molecule FADD, and this delay in apoptosis induction could allow for prolonged cytokine secretion by Hpylori-infected gastric epithelial cells.
基金Supported by the National Institutes of Health Grants DK50669, DK56338 and National Institutes of Health T32 AI007536-06 Training Grant
文摘AIM： TO investigate the role of MHC class Ⅱ in the modulation of gastric epithelial cell apoptosis induced by H pylori infection. METHODS： After stimulating a human gastric epithelial cell line with bacteria or agonist antibodies specific for MHC class Ⅱ and CD95, the quantitation of apoptotic and anti-apoptotic events, including caspase activation, BCL-2 activation, and FADD recruitment, was performed with a fluorometric assay, a cytometric bead array, and confocal microscopy, respectively. RESULTS： Pretreatment of N87 cells with the anti-MHC class Ⅱ IgM antibody RFD1 resulted in a reduction in global caspase activation at 24 h of H pylori infection. When caspase 3 activation was specifically measured, crosslinking of MHC class Ⅱ resulted in a marked reduced caspase activation, while simple ligation of HHC class Ⅱ did not. Crosslinking of HHC class Ⅱ also resulted in an increased activation of the anti-apoptosis molecule BCL-2 compared to simple ligation. Confocal microscope analysis demonstrated that the pretreatment of gastric epithelial cells with a crosslinking anti-HHC class Ⅱ IgH blocked the recruitment of FADD to the cell surface. CONCLUSION： The results presented here demonstrate that the ability of MHC class Ⅱ to modulate gastric epithelial apoptosis is at least partially dependent on its crosslinking. Furthermore, while previous research has demonstrated that MHC class Ⅱ signaling can be proapoptotic during extended ligation, we have shown that the crosslinking of this molecule has anti-apoptotic ef-fects during the earlier time points of Hpylori infection. This effect is possibly mediated by the ability of MHC class Ⅱ to modulate the activation of the pro-apoptotic receptor Fas by blocking the recruitment of the accessory molecule FADD, and this delay in apoptosis induction could allow for prolonged cytokine secretion by Hpyloriinfected gastric epithelial cells.
基金a grant from Pubic Health Ministry of ChinaNatural Science Fundation of Guangdong Province
文摘Purpose:To investigate the density, distribution and morphology of macrophages and MHC class II -positive dendritic cells in the iris and ciliary body of lewis rats. Methods:Immunohistochemistry was performed using monoclonal antibodies specific to monocytes and macrophages (ED1,ED2) and MHC class II -positive cells (OX6) on wholemounts of the iris-ciliary body complex isolated form normal lewis rats.Results:A well developed network of macrophages was present in the iris and ciliary body of normal lewis rats. These cells, morphologically displaying dendriti-form or pleiomorphic appearance, were more densely arranged in mid-iris (950 + 189 cells/mm2) than in iris base (482 ± 78 cells/mm2) and pupil margin (595 ± 92 cells/mm2). A similar network of MHC class II -positive cells with a cell density 452 ± 78 cells/mm2 was almost uniformly distributed in the iris of normal lewis rats.Conclusions : A network of macrophages and MHC class II -positive cells was established in the iris and ciliary body of
基金This study was supported by"948"Project of China(2001-361)Key Project of National Basic Research and De-velopmental Plan(G2000016103)of China.
文摘A fragment spanning over exon 2 and intron 2 of major histocompatibility complex B-LB Ⅱ genes was amplified using PCR, cloned and sequenced in 13 individuals from eight Chinese indigenous chicken breeds and one introduced breed. Another 41 sequences of MHC class Ⅱ β from ten vertebrate species were cited from the NCBI GenBank. Thirteen new B-LB Ⅱ alleles were found in the chicken breeds sampled. Alignment of the exon 2 sequences revealed 91.1-97.8% similarity to each other within the chickens sampled, and the chickens shared 84.1-87.0% homology to Phasianus colchicus, 78.5-81.5% similarity to Coturnix japonica. The sequences in poultry showed 62.6-68.1% identity to HLA-DRB1, 50-61.5% similarity to DQB (HLA-, SLA- and H2-BB), 53.7-60% to HLA-DPB and 53.3-57.8% similarity to HLA-DOB. The frequency of nonsynonymous substitutions of nucleotide was higher than that of synonymous substitutions, and the frequencies of nonsynonymous and synonymous substitutions in poultry B-LB Ⅱ genes were lower than those observed in mammalian DRB1 and DQB1 genes. The deduced amino acid sequences of MHC class Ⅱ β1 domain exhibited extreme difference in conversed region and variable region patterns among the various species, but the two conserved cysteines forming disulfide-bond were shown consistent in poultry with that in mammalian species; and the carbohydrate attachment site was found more conserved in chicken, Homo sapiens, Bos taurus, Ovis aries and Capra hircus than in Sus scrofa and rodent animals. Compared with exon 2 of DQB1 genes of Homo sapiens, ruminant species and Sus scrofa, the differentia that the deletion of six nucleotides at position195 to 200 of exon 2 of DQB1 genes, and insertion of three nucleotides at position 247 to 249 of the exon 2 existed in rodent species were found, which led to the absence of three AA residues at position 65, 66, and 67 within β1 domain of DQB1 chain, and the insertion of one AA residue at position 85. The difference of the deletion of six nucleotides at position 72 to 77 of exon 2 of DPB1 genes was observed with Homo sapiens DQB1, which caused absence of three AA residues at position 24, 25, and 26 of β1 domain of DPB1 chain. The phylogenetic tree revealed that the B-LB Ⅱ sequences from poultry are not orthologous to the class Ⅱ MHC β-chain genes of mammalian species. The tree indicated that genetic evolutionary relationship of chickens with Phasianus colchicus was much closer than with Coturnix japonica, and the DQB and DPB clusters are more tightly related to each other than to the remaining clusters.
基金supported by the National Natural Science Foundation of China(No.31201713,No.31270425 and No.31470442)
文摘The worldwide declines in amphibian populations have largely been caused by infectious fungi and bacteria. Given that vertebrate immunity against these extracellular pathogens is primarily functioned by the major histocompatibility complex（MHC） class Ⅱ molecules, the characterization and the evolution of amphibian MHC class Ⅱ genes have attracted increasing attention. The polymorphism of MHC class Ⅱ genes was found to be correlated with susceptibility to fungal pathogens in many amphibian species, suggesting the importance of studies on MHC class Ⅱ genes for amphibians. However, such studies on MHC class Ⅱ gene evolution have rarely been conducted on amphibians in China. In this study, we chose Omei treefrog（Rhacophorus omeimontis）, which lived moist environments easy for breeding bacteria, to study the polymorphism of its MHC class Ⅱ genes and the underlying evolutionary mechanisms. We amplified the entire MHC class ⅡB exon 2 sequence in the R. omeimontis using newly designed primers. We detected 102 putative alleles in 146 individuals. The number of alleles per individual ranged from one to seven, indicating that there are at least four loci containing MHC class ⅡB genes in R. omeimontis. The allelic polymorphism estimated from the 102 alleles in R. omeimontis was not high compared to that estimated in other anuran species. No significant gene recombination was detected in the 102 MHC class ⅡB exon 2 sequences. In contrast, both gene duplication and balancing selection greatly contributed to the variability in MHC class ⅡB exon 2 sequences of R. omeimontis. This study lays the groundwork for the future researches to comprehensively analyze the evolution of amphibian MHC genes and to assess the role of MHC gene polymorphisms in resistance against extracellular pathogens for amphibians in China.
文摘为了阐明草鱼MHC class I等位基因的结构与多态性,进一步研究其与疾病的关系,从草鱼cDNA文库中克隆了MHC class I基因(Ctid-MHC I);并通过对12个个体Ctid-MHC I的克隆,分析了其等位基因的多态性与三级结构.结果显示Ctid-MHC I等位基因在α1与α2区域变异幅度大,可分为6类(Ctid-MHC I-UA-UF),9型(A-I);但是其三级结构和抗原多肽结合的关键性氨基酸十分保守.结果阐明了草鱼MHC class I分子在8个区域(A-H)置换率高,存在插入或缺失以及长度变异,使等位基因呈现高度多态性.动物MHC class I分子系统树也提示了我国大陆架上鱼类、两栖类、鸟类、哺乳动物和人的遗传距离与分枝年代.
