Olfactomedin-4 regulation by estrogen in the human endometrium requires epidermal growth factor signaling

Olfactomedin-4 (OLFM-4) is an extracellular matrix protein that is highly expressed in human endometrium. We have examined the regulation and function of OLFM-4 in normal endometrium and in cases of endometriosis and endometrial cancer. OLFM-4 expression levels are highest in proliferative-phase endometrium, and 17 beta-estradiol up-regulates OLFM-4 mRNA in endometrial explant cultures. Using the luciferase reporter under control of the OLFM-4 promoter, it was shown that both 17 beta-estradiol and OH-tamoxifen induce luciferase activity, and epidermal growth factor receptor-1 is required for this estrogenic response. In turn, EGF activates the OLFM-4 promoter, and estrogen receptor-alpha is needed for the complete EGF response. The cellular functions of OLFM-4 were examined by its expression in OLFM-4-negative HEK-293 cells, which resulted in decreased vimentin expression and cell adherence as well as increased apoptosis resistance. In cases of endometriosis and endometrial cancer, OLFM-4 expression correlated with the presence of epidermal growth factor receptor-1 and estrogen receptor-alpha (or estrogen signaling). An increase of OLFM-4 mRNA was observed in the endometrium of endometriosis patients. No change in OLFM-4 expression levels were observed in patients with endometrial cancer relative with controts. In conclusion, cross-talk between estrogen and EGF signaling regulates OLFM-4 expression. The role of OLFM-4 in endometrial tissue remodeling before the secretory phase and during the predisposition and early events in endometriosis can be postulated but requires additional investigation. (Am J Pathol 2010, 177:2495-2508: DOI: 10.2353/ajpath.2010.100026

Association of STAT3 and TNFRSF1A with ankylosing spondylitis in Han Chinese

Objectives: Recent association studies by the Australo-Anglo-American Spondyloarthritis Consortium (TASC) in Caucasian European populations from Australia, North America and the UK have identified a number of genes as being associated with ankylosing spondylitis (AS). A candidate gene study in a Han Chinese population was performed based on these findings to identify associated genes in this population. Methods: A case-control study was performed in a Han Chinese population of patients with AS (n=775) and controls (n=1587) from Shanghai and Nanjing. All patients met the modified New York criteria for AS. The cases and controls were genotyped for 115 single nucleotide polymorphisms (SNPs) tagging IL23R, ERAP1, STAT3, JAK2, TNFRSF1A and TRADD, as well as other confirmation SNPs from the TASC study, using the Sequenom iPlex and the ABI OpenArray platforms. Statistical analysis of genotyped SNPs was performed using the Cochran - Armitage test for trend and meta-analysis was performed using METAL. SNPs in AS-associated genes in this study were then imputed using MaCH, and association with AS tested by logistic regression. Results: SNPs in TNFRSF1A (rs4149577, p=8.2×10-4), STAT3 (rs2293152, p=0.0015; rs1053005, p=0.017) and ERAP1 (rs27038, p=0.0091; rs27037, p=0.0092) were significantly associated with AS in Han Chinese. Association was also observed between AS and the intergenic region 2p15 (rs10865331, p=0.023). The lack of association between AS and IL23R in Han Chinese was confirmed (all SNPs p>0.1). Conclusions: The study results demonstrate for the first time that genetic polymorphisms in STAT3, TNFRSF1A and 2p15 are associated with AS in Han Chinese, suggesting common pathogenic mechanisms for the disease in Chinese and Caucasian European populations. Furthermore, previous findings demonstrating that ERAP1, but not IL23R, is associated with AS in Chinese patients were confirmed.

PTHR1 polymorphisms influence BMD variation through effects on the growing skeleton

We investigated whether polymorphisms in PTHR1 are associated with bone mineral density (BMD), to determine whether the association of this gene with BMD was due to effects on attainment of peak bone mass or effects on subsequent bone loss. The PTHR1 gene, including its 14 exons, their exon-intron boundaries, and 1,500 bp of its promoter region, was screened for polymorphisms by denaturing high-performance liquid chromatography (dHPLC) and sequencing in 36 osteoporotic cases. Eleven single-nucleotide polymorphisms (SNPs), one tetranucleotide repeat, and one tetranucleotide deletion were identified. A cohort of 634 families, including 1,236 men (39%) and 1,926 women (61%) ascertained with probands with low BMD (Z< -2.0) and the Children in Focus subset of the Avon Longitudinal Study of Parents and Children (ALSPAC) cohort (785 unrelated individuals, mean age 118 months), were genotyped for the five most informative SNPs (minor allele frequency >5%) and the tetranucleotide repeat. In our osteoporosis families, association was noted between lumbar spine BMD and alleles of a known functional tetranucleotide repeat (U4) in the PTHR1 promoter region (P = 0.042) and between two and three marker haplotypes of PTHR1 polymorphisms with lumbar spine, femoral neck, and total hip BMD (P = 0.021-0.047). This association was restricted to the youngest tertile of the population (age 16-39 years, P = 0.013-0.048). A similar association was found for the ALSPAC cohort: two marker haplotypes of SNPs A48609T and C52813T were associated with height (P = 0.006) and total body less head BMD (P = 0.02), corrected for age and gender, confirming the family findings. These findings suggest a role for PTHR1 variation in determining peak BMD.

Genetics of ankylosing spondylitis

Purpose of Review Over the past 3 years, several new genes and gene deserts have been identified that are associated with ankylosing spondylitis (AS). The purpose of this review is to discuss the major findings of these studies, and the answers they provide and questions they raise about the pathogenesis of this common condition. Recent Findings: Five genes/genetic regions have now definitively been associated with AS [the major histocompatibility complex (MHC), IL23R, ERAP1, 2p15 and 21q22]. Strong evidence to support association with the disease has been demonstrated for the genes IL1R2, ANTXR2, TNFSF15, TNFR1 and a region on chromosome 16q including the gene TRADD. There is an overrepresentation of genes involved in Th17 lymphocyte differentiation/activation among genes associated with AS and the related diseases inflammatory bowel disease and psoriasis, pointing strongly to this pathway as playing a major causative role in the disease. Increasing information about differential association of HLA-B27 subtypes with disease suggests a hierarchy of strength of association of those alleles with AS, providing a useful test as to the validity of different potential mechanisms of association of HLA-B27 with AS. The mechanism underlying the association of the gene deserts, 2p15 and 21q22, suggests the involvement of noncoding RNA in AS etiopathogenesis. Summary: The increasing list of genes identified as being definitely involved in AS provides a useful platform for hypothesis-driven research in the field, providing a potential alternative route to determining the underlying mechanisms involved in the disease to research focusing on HLA-B27 alone.

Genetic determinants of bone density and fracture risk: State of the art and future directions

Context: Osteoporosis is a common, highly heritable condition that causes substantial morbidity and mortality, the etiopathogenesis of which is poorly understood. Genetic studies are making increasingly rapid progress in identifying the genes involved. Evidence Acquisition and Synthesis: In this review, we will summarize the current understanding of the genetics of osteoporosis based on publications from PubMed from the year 1987 onward. Conclusions: Most genes involved in osteoporosis identified to date encode components of known pathways involved in bone synthesis or resorption, but as the field progresses, new pathways are being identified. Only a small proportion of the total genetic variation involved in osteoporosis has been identified, and new approaches will be required to identify most of the remaining genes.

Breakthroughs in genetic studies of ankylosing spondylitis

Ankylosing spondylitis (AS), the prototypic seronegative arthropathy, is known to be highly heritable, with >90% of the risk of developing the disease determined genetically. As with most common heritable diseases, progress in identifying the genes involved using family-based or candidate gene approaches has been slow. The recent development of the genome-wide association study approach has revolutionized genetic studies of such diseases. Early studies in ankylosing spondylitis have produced two major breakthroughs in the identification of genes contributing roughly one third of the population attributable risk of the disease, and pointing directly to a potential therapy. These exciting findings highlight the potential of future more comprehensive genetic studies of determinants of disease risk and clinical manifestations, and are the biggest advance in our understanding of the causation of the disease since the discovery of the association with HLA-B27.

Variation in BMPR1B, TGFRB1 and BMPR2 and control of dizygotic twinning

Genes in the TGF9 signaling pathway play important roles in the regulation of ovarian follicle growth and ovulation rate. Mutations in three genes in this pathway, growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15) and the bone morphogenetic protein receptor B 1 (BMPRB1), influence dizygotic (DZ) twinning rates in sheep. To date, only variants in GDF9 and BMP15, but not their receptors transforming growth factor ss receptor 1 (TGFBR1), bone morphogenetic protein receptor 2 (BMPR2) and BMPR1B, have been investigated with respect to their roles in human DZ twinning. We screened for rare and novel variants in TGFBR1, BMPR2 and BMPR1B in mothers of dizygotic twins (MODZT) from twin-dense families, and assessed association between genotyped and imputed variants and DZ twinning in another large sample of MODZT. Three novel variants were found: a deep intronic variant in BMPR2, and one intronic and one non-synonymous exonic variant in BMPRB1 which would result in the replacement of glutamine by glutamic acid at amino acid position 294 (p.Gln294Glu). None of these variants were predicted to have major impacts on gene function. However, the p.Gln294Glu variant changes the same amino acid as a sheep BMPR1B functional variant and may have functional consequences. Six BMPR1B variants were marginally associated with DZ twinning in the larger case-control sample, but these were no longer significant once multiple testing was taken into account. Our results suggest that variation in the TGF9 signaling pathway type II receptors has limited effects on DZ twinning rates in humans.

Cytokinin signalling in the regulation of cambial development

Secondary growth of plants is of pivotal importance in terrestrial ecosystems, providing a significant carbon sink in the form of wood. As plant biomass accumulation results largely from the cambial growth, it is surprising that quite little is known about the hormonal or genetic control of this important process in any plant species. The central aim of my thesis studies was to explore the function of cytokinin in the regulation of cambial development. Since their discovery as regulators of plant cell divisions, cytokinins have been assumed to participate in the control of cambial development. Evidence for this action was deduced from hormone treatment experiments, where exogenously applied cytokinin was shown to enhance cambial cell divisions in diverse plant organs and species. In my thesis work, the conservation of cytokinin signalling and homeostasis genes between a herbaceous plant, Arabidopsis, and a hardwood tree species, Populus trichocarpa. Presumably reflecting the ancient origin of cytokinin signalling system, the Populus genome contains orthologs for all Arabidopsis cytokinin signalling and homeostasis genes. Thus, genes belonging to five main families of isopentenyl transferases (IPTs), cytokinin oxidases (CKXs), two-component receptors, histidine containing phosphotransmitters (HPts) and response regulators (RRs) were identified from the Populus genome. Three subfamilies associated with cytokinin signal transduction, the CKI1-like family of two-component receptors, the AHP4-like HPts, and the ARR22-like atypical RRs, were significantly larger in Populus genome than in Arabidopsis. Potential contribution to the extensive secondary development of Populus by the members of these considerably expanded gene families will be discussed. Representatives of all cytokinin signal transduction elements were expressed in the Populus cambial zone, and most of the expressed genes appeared to be slightly more abundant on the phloem side of the meristem. The abundance of cytokinin related genes in the cambium emphasizes the important role of this hormone in the regulation of the extensive secondary growth characteristic of tree species. The function of the pseudo HPts in primary vascular development was studied in Arabidopsis root vasculature. It was demonstrated that the pseudo HPt AHP6 has a role in locally inhibiting cytokinin signalling in the protoxylem position in the Arabidopsis root, thus enabling differentiation of the protoxylem cell file. The possible role of pseudo HPts in cambial development will be discussed. The expression peak of cytokinin signalling genes in the tree cambial zone strongly indicates that cytokinin has a role in the regulation of this meristem function. To address whether cytokinin signalling is required for cambial activity, transgenic Populus trees with modified cytokinin signalling were produced. These trees were expressing a cytokinin catabolic gene from Arabidopsis, CYTOKININ OXIDASE 2, (AtCKX2) under the promoter of a Betula CYTOKININ RECEPTOR 1 (BpCRE1). The pBpCRE1::CKX2 transgenic Populus trees showed a reduced concentration of a biologically active cytokinin, correlating with their impaired cytokinin response. Furthermore, the radial growth of these trees was compromised, as illustrated by a smaller stem diameter than in wild-type trees of the same height. Moreover, the level of cambial cytokinin signalling was down-regulated in these thin-stemmed trees. The reduced signalling correlated with a decreased number of meristematic cambial cells, implicating cytokinin activity as a direct regulator of cambial cell division activity. Together, the results of my study indicate that cytokinins are major hormonal regulators required for cambial development.

FGFR1 regulated gene-expression, cell proliferation and differentiation in the developing midbrain and hindbrain

The neuroectodermal tissue close to the midbrain hindbrain boundary (MHB) is an important secondary organizer in the developing neural tube. This so-called isthmic organizer (IsO) regulates cellular survival, patterning and proliferation in the midbrain (Mb) and rhombomere 1 (R1) of the hindbrain. Signaling molecules of the IsO, such as fibroblast growth factor 8 (FGF8) and WNT1 are expressed in distinct bands of cells around the MHB. It has been previously shown that FGF-receptor 1 (FGFR1) is required for the normal development of this brain region in the mouse embryo. In the present study, we have compared the gene expression profiles of wild-type and Fgfr1 mutant embryos. We show that the loss of Fgfr1 results in the downregulation of several genes expressed close to the MHB and in the disappearance of gene expression gradients in the midbrain and R1. Our microarray screen identified several previously uncharacterized genes which may participate in the development of midbrain R1 region. Our results also show altered neurogenesis in the midbrain and R1 of the Fgfr1 mutants. Interestingly, the neuronal progenitors in midbrain and R1 show different responses to the loss of signaling through FGFR1. As Wnt1 expression at the MHB region requires the FGF signaling pathway, WNT target genes, including Drapc1, were also identified in our screen. The microarray data analysis also suggested that the cells next to the midbrain hindbrain boundary express distinct cell cycle regulators. We showed that the cells close to the border appeared to have unique features. These cells proliferate less rapidly than the surrounding cells. Unlike the cells further away from the boundary, these cells express Fgfr1 but not the other FGF receptors. The slowly proliferating boundary cells are necessary for development of the characteristic isthmic constriction. They may also contribute to compartmentalization of this brain region.

Myoblast Sheet Transplantation for Treatment of Heart Failure after Myocardial Infarction

Heart failure is a common, severe, and progressive condition associated with high mortality and morbidity. Because of population-aging in the coming decades, heart failure is estimated to reach epidemic proportions. Current medical and surgical treatments have reduced mortality, but the prognosis for patients has remained poor. Transplantation of skeletal myoblasts has raised hope of regenerating the failing heart and compensating for lost cardiac contractile tissue. In the present work, we studied epicardial transplantation of tissue-engineered myoblast sheets for treatment of heart failure. We employed a rat model of myocardial infarction-induced acute and chronic heart failure by left anterior descending coronary artery ligation. We then transplanted myoblast sheets genetically modified to resist cell death after transplantation by expressing antiapoptotic gene bcl2. In addition, we evaluated the regenerative capacity of myoblast sheets expressing the cardioprotective cytokine hepatocyte growth factor in a rat chronic heart failure model. Furthermore, we utilized in vitro cardiomyocyte and endothelial cell culture models as well as microarray gene expression analysis to elucidate molecular mechanisms mediating the therapeutic effects of myoblast sheet transplantation. Our results demonstrate that Bcl2-expression prolonged myoblast sheet survival in rat hearts after transplantation and induced secretion of cardioprotective, proangiogenic cytokines. After acute myocardial infarction, these sheets attenuated left ventricular dysfunction and myocardial damage, and they induced therapeutic angiogenesis. In the chronic heart failure model, inhibition of graft apoptosis by Bcl-2 improved cardiac function, supported survival of cardiomyocytes in the infarcted area, and induced angiogenesis in a vascular endothelial growth factor receptor 1- and 2-dependent mechanism. Hepatocyte growth factor-secreting myoblast sheets further enhanced the angiogenic efficacy of myoblast sheet therapy. Moreover, myoblast-secreted paracrine factors protected cardiomyocytes against oxidative stress in an epidermal growth factor receptor- and c-Met dependent manner. This protection was associated with induction of antioxidative genes and activation of the unfolded protein response. Our results provide evidence that inhibiting myoblast sheet apoptosis can enhance the sheets efficacy for treating heart failure after acute and chronic myocardial infarction. Furthermore, we show that myoblast sheets can serve as vehicles for delivery of growth factors, and induce therapeutic angiogenesis in the chronically ischemic heart. Finally, myoblasts induce, in a paracine manner, a cardiomyocyte-protective response against oxidative stress. Our study elucidates novel mechanisms of myoblast transplantation therapy, and suggests effective means to improve this therapy for the benefit of the heart failure patient.

Administração crônica de cafeína durante a gestação afeta o remodelamento cardíaco e expressão dos componentes do sistema renina angiotensina da prole adulta de camundongos C57BL/6

Este estudo teve como objetivo avaliar o papel da administração crônica de cafeína durante a gestação de camundongos C57BL/6 sobre o remodelamento cardíaco e a expressão de componentes do sistema renina-angiotensina (SRA) na prole macho adulta. Fêmeas C57BL/6 grávidas foram divididas em dois grupos (n = 10): grupo controle (C), progenitoras foram injetadas apenas com o veículo (solução salina NaCl 0,9%), e grupo cafeína (CF), progenitoras receberam diariamente uma injecção subcutânea contendo 20 mg/kg de cafeína (1 mg/ml de solução salina). Após o desmame, os filhotes tiveram livre acesso à ração padrão até 90 dias de idade quando foram sacrificados. Rim e ventrículo esquerdo (VE) foram coletados para análise estrutural e western blotting. O grupo cafeína mostrou uma redução significativa no ganho de massa corporal (MC) (-18%; P <0,0001). O grupo cafeína apresentou ainda um aumento da pressão arterial sistólica (+ 48%; P <0,0001) e freqüência cardíaca (+10%; P <0,01) em relação ao grupo controle. A massa do VE corrigida pela MC no grupo da cafeína foi maior que no grupo C (+10%; P <0,01). O grupo cafeína apresentou um aumento na área de cardiomiócitos (+40%; P <0,05), e reduzida densidade capilar (-25%; P <0,05). No rim, as expressões de renina (128%; P <0,05) e dos receptores 1 da angiotensina II (AT1R) (88%; P <0,05) foram significativamente maiores nos animais do grupo cafeína. No VE, o grupo cafeína demonstrou aumento da expressão de ECA (+30%; P <0,05), angiotensina II (+60%; P <0,01), e AT1R (+77%; P <0,01) e diminuição da expressão do receptor 2 de angiotensina II (-46%; P <0,05). Em conclusão, a administração crônica de cafeína durante a gestação, possivelmente programa a expressão de componentes de sistema renina-angiotensina, levando à ativação persistente do SRA renal e cardíaco local, que por sua vez promove o aumento da pressão sanguínea, remodelação e efeitos cardíacos adversos.

Efeitos da exposição à nicotina e à fumaça de cigarro durante a lactação sobre o sistema de recompensa cerebral em ratos

Crianças de mães fumantes são mais suscetíveis a se tornarem adultos obesos e se viciarem em drogas ou alimentos palatáveis. Drogas e alimentos ativam a via mesolímbica de recompensa, causando sensação de prazer que induz ainda mais o consumo. Assim, avaliamos a relação entre a exposição apenas à nicotina ou à fumaça do cigarro durante a lactação com a preferência alimentar e sistema dopaminérgico de recompensa cerebral das proles, em dois modelos de programação: Modelo I: no 2o dia pós-natal (PN), lactantes receberam implante de minibombas osmóticas que liberam nicotina (NIC) ou salina (C), durante 14 dias. Em PN150 e novamente em PN160, as proles foram divididas em 4 grupos para um desafio alimentar: N-SC e C-SC que receberam ração padrão; N-SSD e C-SSD que podiam escolher livremente entre as dietas hiperlipídica e hiperglicídica. A ingestão alimentar foi avaliada após 12 h. As mães foram sacrificadas apenas na 21 da lactação (desmame) e as proles em PN15 (com nicotina), PN21 e PN170 (ausência da NIC). Ao desmame, as ratas lactantes NIC apresentaram menor conteúdo de tirosina hidroxilase (TH), maior OBRb e SOCS3 na area tegmentar ventral (VTA); menor TH, maior receptor de dopamina 1 (D1R), receptor de dopamina 2 (D2R) e transportador de dopamina (DAT) no núcleo accumbens (NAc); maior conteúdo de TH no estriado dorsal (DS); e maior D2R e SOCS3 no núcleo arqueado (ARC). Em PN15, os filhotes NIC apresentaram maior conteúdo de D1R, D2R e menor DAT no NAc, enquanto em PN21, apresentaram apenas menor DAT no DS, e menor conteúdo de pSTAT3 em ARC. Aos 170 dias, as proles SSD demonstraram maior preferência para a ração hiperlipídica. No entanto, os animais N-SSD consumiram mais ração hiperglicidica do que as proles C-SSD. A prole N apresentou menor conteúdo de D2R e DAT no NAc e menor D2R no ARC. Modelo II: as mães e suas proles foram divididas em: expostos à fumaça do cigarro (grupo S: 4 vezes / dia, do 3 ao 21 dia de lactação), e expostos ao ar filtrado (grupo C). Em PN175, as proles foram divididas em 4 grupos para o desafio alimentar S-SC, C-SC, S-SSD e C-SSD. A ingestão alimentar foi avaliada após 30 min e 12 h. Em PN180, as proles foram sacrificadas. O grupo S-SSD ingeriu mais das rações palatáveis do que o grupo C-SSD em 30 min e 12 h. Ambos os grupos preferiram a ração hiperlipídica. No entanto, os animais S-SSD consumiram mais ração hiperlipídica do que C-SSD em 30 min. A prole S apresentou menor conteúdo de TH no VTA, menor conteúdo de TH, D2R e maior conteúdo de D1R no NAc e menor OBRb no ARC. Demonstramos que tanto a nicotina isolada como a exposição à fumaça do cigarro durante a lactação resultaram em mudanças no sistema dopaminérgico das proles, programando o comportamento alimentar devido à diminuição da dopamina no NAc.

兔胚胎干细胞分离建系及鉴定以及胚胎干细胞自我更新机制研究

人胚胎干细胞（ESC）的成功分离培养，吸引大批人对干细胞生物学的关注，特 别是ESC 在再生医学及人类早期胚胎发育研究的潜在价值。然而在人ESC 临床应用 之前需要找到合适的动物模型进行大量的预实验研究，从而评价其应用的安全性、有 效性及存活效率。因此，从其它物种建立稳定而可用的ESC 系也是必不可少的。ESC 能无限地自我更新并保持多潜能性，但控制其自我更新的分子机制现在仍然知之甚少 且物种间存在差异，了解ESC 的自我更新有利于提高建系率、改善培养体系及定向 分化体系。本文一方面对ESC 分离培养及自我更新机制的研究进展进行了综述，另 一方面对以下几个方面的内容进行了研究：1）建立了4 株稳定的兔ESC 系，能在体 外进行长期的培养并保持ESC 的多潜能Markers 及正常的XY 或XX 核型，具有碱性 磷酸酶活性、表达Oct-4、SSEA-1、SSEA-3、SSEA-4、TRA-1-60 及TRA-1-81。与人 和小鼠ESC 相似，兔ESC 表达多潜能基因（Oct-4、Nanog、Sox-2 及UTF-1），并表 达了与ESC 自我更新相关的信号通路（FGF、TGFβ及WNT）的许多基因。从形态 来说，兔ESC 与灵长类ESC 相似，但兔ESC 具有较快的增殖能力，与小鼠ESC 相类 似。在体外及体内兔ESC 均能分化成代表原始三胚层的各种细胞类型及组织。2) 从 受体抑制实验及生长因子的联合加入可以得出结论FGF 及TGF 信号通路对维持兔 ESC 的多潜能性发挥着重要的作用，这样的结果与人ESC 相类似。也表明FGF、TGF β及WNT 信号通路在兔ESC 的自我更新中都起着作用，而且他们之间可能形成了信 号调控网络，相互之间有着正负反馈作用。FGF2+Activin A 或TGFβ1+Noggin 的无 饲养层无血清培养体系不仅能显著抑制兔ESC 的分化，且能维持其长期的自我更新。 但与小鼠不同，TGFβ信号通路能影响其增殖能力，而对其多潜能性的维持并没有作 用。这就更说明了兔比小鼠更适宜成为人类疾病临床治疗之前的模型动物。3）四种 猕猴细胞系（MOF、MESF、MFG 和CMESF）可作为饲养层比MEFs（小鼠饲养层 细胞）更好或同等好支持猕猴ESC 的生长，保持其自我更新能力和分化的多能性。 而卵泡颗粒上皮样细胞（MFGE）不能支持猕猴ESC 的自我更新。进一步的研究表明 饲养层支持ESC 生长能力的差异可能是由于基因表达种类以及表达量上的差异而导 致的。

苯肾上腺素诱导乳鼠心肌细胞蛋白质表达变化的蛋白质组分析

α1-肾上腺素受体(α1-Adrenergic receptor,α1-AR)是G蛋白偶联受体(G-protein coupled receptor,GPCR),也是内源性儿茶酚胺、去甲肾上腺素和肾上腺素最重要的靶受体之一.α1-AR广泛分布于机体的各种器官、组织和细胞中,并介导多种生理效应,如血管收缩、蛋白质合成及心脏变力变时作用等[1,2].很多研究已经证实,α1-AR及其信号转导通路与许多心血管疾病存在密切关系[3,4].蛋白质组学可提供一种发现在疾病情况下异常表达蛋白质的方法,为疾病的早期诊断和愈后判断提供指南,并为针对性疾病治疗提供科学依据.本研究以乳鼠心肌细胞为实验模型,利用双向凝胶电泳和飞行时间质谱分析苯肾上腺素诱导乳鼠心肌细胞表达变化的蛋白质.1实验部分1.1试剂苯肾上腺素(Phenylephine,PE)购自Sigma公司;胰蛋白酶和DMEM购自Hyclone公司;IPG预制胶条(pH=5~8,胶条长17 cm),载体两性电解质(B io-Lyte5-8)购于B io-Rad公司;TPCK修饰的测序级胰酶购自Promega公司;其它试剂均为国产分析纯.1.2实验过程(1)乳鼠心肌细胞培养及样...

调控文蛤幼虫变态的肾上腺素能受体类型及幼虫发育中神经和肌肉的变化

大部分贝类幼虫在发育过中要经历从浮游生活到底栖生活的变化过程，同时形态结构也要经历巨大的变化，这个过程称为变态。 变态是文蛤幼虫发育过程中非常重要的一个阶段。 药理学和细胞免疫学证据表明β肾上腺素样受体在文蛤幼虫变态过程中有重要作用。药理学实验分别采用了几种儿茶酚胺类受体的激动剂和抑制剂来处理幼虫，检验它们在幼虫变态过程中的作用。结果表明，在10μM和100μM的浓度下，肾上腺素(AD)和去甲肾上腺素(NA)中能够显著提高幼虫的变态率(p<0.05)。10μM和100μM浓度的AD能够提高幼虫变态率30%左右。10μM和100μM浓度的NA能分别提高幼虫变态率35.3%和27.6%。10μM的β受体激动剂－isoproterenol也能够显著的提高幼虫的变态率30%(p<0.05)，但是α受体激动剂－phenylephrine在0.1μM到100μM的浓度范围内不能显著提高幼虫的变态率(p>0.05)。而且，1μM的β受体抑制剂－propanolol能显著的抑制AD或NA提高幼虫变态率的作用(p<0.05)；但是α受体抑制剂－prazosin对AD或NA提高幼虫变态率没有显著性影响(p>0.05)。 此外本文还利用整装免疫细胞化学的方法进一步研究了文蛤幼虫不同发育阶段，神经系统和β肾上腺素样受体的发育情况。 幼虫的神经系统在担轮幼虫时期(受精后18h)开始发育，这时还不能检测到β肾上腺素受体。面盘幼虫时期(受精后1d)具备了顶神经节、脑神经节和脏神经节组成的中枢神经系统，在口附近有一些外周神经。β肾上腺素受体在受精后24h首次出现在面盘幼虫的顶神经节和脑神经节，分别命名为AR(apical receptor)和CR1(cerebral receptor 1)。 在受精后5d顶神经节已经检测不到。脑神经节和脏神经节由腹部向背部迁移，口的背腹两侧都出现了一些神经元。并且脏神经节周围也出现了一些神经细胞。AR在受精后3d就检测不到了。同时在CR1的后部新出现了一些β肾上腺素受体，命名为CR2。此后CR2发育迅速，在受精后5d就和CR1差不多大小。并且在CR1和CR2之间还出现了很多小的阳性信号。 变态过程中中枢神经系统中顶神经节消失了，并且出现了足神经节。另外，外套膜上出现了更多的外周神经。除了在稚贝的脑神经节和脏神经节外，在足、外套膜和水管上都有β肾上腺素受体存在。成体的鳃、足、心脏、水管、唇掰和外套膜上也有β肾上腺素受体分布。 变态信号传递到靶器官后，文蛤幼虫在形态结构和生态习性上开始了快速巨大的变化。其中幼虫的肌肉系统是变化最大的系统，并且直接与文蛤运动、摄食等习性转变相关。本文采用免疫组化的方法对文蛤幼虫肌肉系统的发育进行了研究。结果表明，文蛤幼虫具有由幼虫收缩肌、面盘收缩肌和闭壳肌组成的十分复杂的肌肉系统。幼虫收缩肌和前闭壳肌最早出现于担轮幼虫阶段(受精后18h)。孵化后幼虫的肌肉系统迅速发育，受精后22h幼虫的肌肉系统基本发育完全，并一直维持到变态前。变态过程中幼虫收缩肌和面盘收缩肌逐渐萎缩消失。同时，足收缩肌、后闭壳肌和外套膜肌肉等稚贝的肌肉系统快速形成。变态后稚贝幼虫收缩肌和面盘收缩肌完全消失，其肌肉系统由前后闭壳肌、足收缩肌和外套膜肌肉组成。