神经营养素3和受体在人胚脊髓发育中的定位表达

目的了解神经营养素3(NT-3)和受体TrkC在人胚脊髓发育过程中的定位分布。方法用4、5、6周龄3个时段的人胚进行免疫组织化学ABC法染色以了解不同时段NT-3和受体在人胚脊髓发育中的定位表达和分布情况。结果在人胚胎脊髓发育的3个实验段,NT-3阳性颗粒主要分布于胞质,其受体TrkC主要分布于胞膜;发育过程中NT-3及其受体在室管膜层-套层-边缘层3条带上呈现不同程度的免疫阳性反应。结论NT-3及其受体TrkC在不同时期的人胚胎脊髓发育中进行表达,提示NT-3可能诱导神经干细胞分裂增殖,并协同其它生长因子促进轴突的生长,保证神经元胞体的存活。     

脑源性神经营养因子在早期人胚神经管发育中的定位表达

目的研究脑源性神经营养因子(BDNF)在早期人胚神经管发育过程中的定位表达。方法采用免疫细胞化学ABC法染色,研究35天人胚的发育情况。结果在人胚神经管的室带中,神经元的细胞质BDNF免疫反应阳性;在中间带,神经元的突起BDNF免疫反应阳性,一部分神经元的细胞核BDNF免疫反应阳性,另外一部分神经元的细胞核BDNF免疫反应阴性;在缘带BDNF的分布与中间带相似。神经管的头侧较尾侧BDNF阳性反应较强,神经管的腹侧BDNF阳性反应较背侧强。结论BDNF在人胚神经管免疫反应阳性,表明BDNF是诱导神经管分化发育的重要信号分子,提示BDNF在人胚神经管的发育中具有十分重要的作用。     

神经营养素-3的研究进展

神经营养素－３（ｎｅｕｒｏｔｒｏｐｈｉｎ－３，ＮＴ－３）是神经生长因子基因家族的成员，可促进多种中枢和外周神经元的存活和分化，调节神经元突触活动，并对神经系统发育和成熟起重要作用。ＮＴ－３在损伤条件下对神经元具有保护作用，因而在治疗神经系统疾病和神经损伤中有临床应用前景     

人胚早期心脏发育过程中VEGF-C及其受体的表达

目的：观察人胚早期心脏VEGF-C及其受体VEGFR-2、VEGFR-3的表达变化，探讨其在早期心脏发育过程中的作用。方法：收集第3-8周人胚，石蜡切片，免疫组化染色，光镜观察，分析心脏组织发育中VEGF-C及其受体的表达变化、心脏组织的分化和形成。结果：VEGF-C、VEGFR-2、VEGFR-3在心内膜内皮细胞及心肌细胞发育第3、4周开始表达，随胎龄增加表达先增强后逐渐减弱，第7周末基本消失。结论：VEGF-C及其受体参与了人胚早期心脏的形成过程，对早期心脏发育发挥了一定的作用。     

神经营养素受体在幼年大鼠神经系统表达的特点

为了研究神经营养素受体 ( Trks)在幼年大鼠神经系统的表达模式及分布规律。本研究应用免疫组织化学技术对幼年大鼠大脑皮质、小脑、脊髓、背根神经节中 Trks的表达及分布进行了形态学观察及定位。结果证明 ,神经营养素受体 Trk A,Trk B,Trk C在大脑皮质、脊髓、背根神经节中均有表达 ,Trk B呈强阳性且分布广 ,Trk A次之 ,Trk C为弱阳性 ;在小脑 Purkinje细胞Trk C表达阳性 ,Trk A弱阳性 ,Trk B阴性。结论 :幼年大鼠神经系统中 Trks表达存在差异 ,提示神经系统不同部位发育所需的神经营养素不同 ;Trks的分布部位与已报道的神经营养素的分布部位不完全重叠 ,可能与神经营养素作用模式多样性有关     

人神经营养素-3全基因在大肠杆菌内的表达

目的　应用基因工程方法制备人神经营养素 3(hNT 3)蛋白 ,为研究hNT 3的生物学作用和开展应用hNT 3进行中枢神经损伤和退行性疾病的治疗提供材料。方法　将经过序列分析确定的hNT 3全基因重组至原核表达质粒pBV2 2 0中 ,构建了重组表达载体pBV/hNT 3,在大肠杆菌中表达后 ,SDS PAGE法测定目的蛋白的相对分子质量 ,鸡胚背根节培养法检测其生物学活性。结果　pBV/hNT 3在大肠杆菌中表达出了三个分子质量分别为 1 5× 1 0 3、1 8× 1 0 3和 33× 1 0 3的蛋白质 ,恰与hNT 3蛋白、其前导肽及前体的分子量相符。表达蛋白主要以包涵体形式存在 ,经纯化复性后 ,可明显促进鸡胚背根节的存活和神经元突起的生长。结论　人神经营养素 3全基因可在大肠杆菌内正确地转录和翻译 ,并可部分性地进行加工     

神经营养因子在早期人胚神经管发育中的定位表达

目的尽管在体外细胞培养实验中已经证实纤维细胞生长因子（FGF）、胰岛素样生长因子（IGF）、血管内皮生长因子（EGF）等因子具有神经营养作用,但是它们在神经管发育过程中的定位表达和变化规律仍然还不清楚,因此本实验将探索神经营养因子在人胚发育过程中的表达及意义。方法用23 d、45 d和56 d的人胚进行免疫组织化学ABC法染色,分析FGF、IGF、EGF在神经发育过程中的表达及意义。结果在早期人胚神经管发育中,室管膜层、中间层、边缘层均可见IGF、FGF、EGF表达,各层细胞胞质及细胞之间可见棕黄色免疫反应阳性物。结论在人胚早期发育的过程中,IGF起主导作用,EGF和FGF起协同作用,共同促进神经管的分化发育。     

人神经营养素-3基因真核表达载体的构建和鉴定

目的：构建含有人NT-3基因真核表达载体pcDNA3．1-NT-3。方法：采用PCR技术，扩增编码神经营养素-3基因，克隆到PMD18-T载体上，再亚克隆到真核表达载体pcDNA3．1的Hind Ⅲ和BamHI位点。结果：重组真核表达载体pcDNA3．1-NT-3经酶切鉴定证明NT-3基因正向插入真核表达载体中，碱基序列的测定证明重组质粒中含有人的NT-3基因序列。结论：构建的真核表达载体携有人NT-3cDNA序列，并且含有巨细胞病毒强启动子、ploy(A)加尾信号和neo标志基因，可能具有转染多种哺乳类细胞通用性，可用于NT-3基因的真核表达及基因治疗的研究。     

人胚早期心脏发育过程中VEGF及其受体的表达

目的观察人胚早期心脏VEGF-A和VEGF-C及其受体VEGFR-1、VEGFR-2、VEGFR-3的表达变化,探讨VEGF-A和VEGF-C在早期心脏发育过程中的作用。方法收集第3～8周人胚,石蜡切片,免疫组化染色,光镜观察,分析心脏组织发育中VEGF家族及其相关受体的表达变化、心脏组织的分化和形成。结果VEGF-A、VEGF-C、VEGFR-1、VEGFR-2、VEGFR-3在心内膜内皮细胞及心肌细胞发育第3、4周开始表达,随胎龄增加表达先增强后逐渐减弱,第7周左右消失。结论VEGF-A和VEGF—C参与了人胚早期心脏的形成过程,对早期心脏发育发挥了一定的作用。     

督脉电针对早期受损伤的脊髓神经营养素-3表达的影响

目的 观察督脉电针对早期受损伤的脊髓神经营养素-3(NT-3)表达的影响及其细胞定位.方法 成年雌性大鼠分为损伤组和电针加损伤组.全横断损伤两组大鼠的脊髓1d后,开始对电针+损伤组大鼠进行督脉电针,损伤组大鼠不做督脉电针.电针加损伤组在电针后1、3和7d时间点取出脊髓损伤区组织,损伤组也在相应时间点取出损伤区组织,用ELISA方法检测损伤区组织NT-3水平.再取电针后7d大鼠脊髓损伤区及其邻近组织切片做NT-3的免疫荧光组织化学双标染色.结果 电针加损伤组和损伤组的脊髓损伤区组织NT-3表达在时间上基本一致;在前3 d,NT-3水平是下降的,在后3 d,NT-3水平是增高的.但是,与损伤组相比,电针加损伤组的NT-3水平是明显增高(P＜0.05).免疫荧光双标染色结果显示,神经元、星形胶质细胞、少突胶质细胞和巨噬细胞都有NT-3的表达.结论 督脉电针可以促进受损伤早期的脊髓组织细胞合成和分泌内源性NT-3,这可能是督脉电针促进急性脊髓损伤修复的适宜微环境因素之一.     

人胚胎脊髓发育过程中神经营养因子3的表达及变化

目的:探讨神经营养因子3(NT-3)在不同发育时期人胚胎脊髓中的表达变化及意义.方法:采用免疫组织化学和免疫印迹法对4周～7月人胚胎脊髓发育过程中NT-3的定位及定量进行研究.结果:NT-3在所检测的11个时段中均有表达,从第6周开始逐渐增加,至第9周时达高峰,之后下降并维持在一定水平,且以细胞突起和纤维表达最明显.结...     

Expression of cystic fibrosis transmembrane conductance regulator during early human embryo development

Formation of the blastocyst is one of the first morphological changes in early embryonic development. Ion transport has been shown to be crucial for blastocoele cavity formation and expansion, although the mechanisms that underlie this process are presently unknown. As a transmembrane Cl(-) channel, the cystic fibrosis transmembrane conductance regulator (CFTR) may participate in ion transport and early blastocoele formation. CFTR mRNA was detected throughout preimplantation embryo development and in the unfertilized oocyte. Immunocytochemistry disclosed the presence of CFTR protein from the 8-cell stage, reaching maximum immunoreactivity at early blastocyst stage embryos. Patch clamp electrophysiology of morulae and blastocysts demonstrated typical CFTR Cl(-) channel activities in the apical membrane of trophectoderm cells. Thus CFTR is expressed both at mRNA and protein levels in human morulae and blastocysts, and functions as a cAMP-regulated apical membrane Cl(-) channel. These data suggest that CFTR may contribute to blastocoele formation in the early human embryo.     

Expression of intercellular junctions during preimplantation development of the human embryo

A total of 74 human embryos were stained with gap junction proteinspecific anti-peptide antibodies and antibodies to the desmosomalprotein desmoplakin to reveal the expression pattern of intercellularjunctions during preimplantation development. Prior to implantation,the human embryo expresses predominantly connexin (Cx43)-containinggap junctions. Gap junctions were first detected in apposingcell membranes at the 4-cell stage and became increasingly organizedas development proceeded. In normal blastocysts, trophectoderm(TE) cells were linked by dense arrays of gap junctions whileinner cell mass (ICM) cells were linked by small, punctate gapjunctions. Gap junctions containing Cx32 or Cx26 were observedoccasionally in the TE of late blastocysts. Desmosomes appearedbetween outer cells prior to cavitation and were retained inthe TE, but not in the ICM. Levels of gap junction protein expressionwere variable in morphologically normal embryos at the samestage, suggesting that a normal appearance may not be a reliableindicator of future viability. Morphologically normal embryosoften possessed multinucleate, apoptotic and decompacting cells.They could show either extensive, disorganized over-expressionor reduced expression of gap junction protein. The results fitthe view that only embryos destined to survive display an organizedpattern of intercellular junctions. blastocyst/connexin/desmosome/gap junction/immunocytochemistry     

A role for p75 receptor in neurotrophin-3 functioning during the development of limb proprioception

Neurotrophin-3 is indispensable for the development of limb proprioceptive neurons and their end organs, muscle spindles. To determine whether the low-affinity p75 receptor potentiates the actions of neurotrophin-3, we examined the development of the proprioceptive system in p75 null mutant mice that had either normal or decreased tissue levels of neurotrophin-3. Postnatal mice lacking both copies of the p75 gene had fewer sensory neurons in dorsal root ganglia, but normal complements of muscle spindles in fast hindlimb muscles, although the slow soleus muscle showed a 50% loss of spindles. However, compound mutants lacking both copies of the p75 gene as well as one copy of the neurotrophin-3 gene displayed a dystonic/ataxic phenotype similar to that observed previously in neurotrophin-3 null mutants devoid of proprioception. The compound mutants also exhibited a commensurate loss of parvalbumin-expressing (proprioceptive) neurons in dorsal root ganglia. The degree of deficiency of spindles (and presumably proprioceptive neurons) in the compound mutants exceeded the sum of deficits in single mutants lacking either both copies of p75 genes or one copy of neurotrophin-3 gene, suggesting a synergistic interaction between the p75 receptor and neurotrophin-3. Neuronal deficits in the compound mutants were present prior to embryonic day 14, indicating an early role for the p75 receptor in sensory neuronogenesis. Collectively, these data indicate that the p75 receptor is not essential for the survival and differentiation of most limb proprioceptive neurons when neurotrophin-3 is expressed at normal levels. However, the p75 receptor may act in synergy with neurotrophin-3 to enhance the survival of proprioceptive neurons when tissue levels of neurotrophin-3 are a limiting factor.     

Expression of cell adhesion molecules during human preimplantation embryo development

Formation of a fully differentiated, implantation competent blastocyst requires the expression of a complex repertoire of molecules. However, the events that drive morphogenesis are poorly elucidated in the human embryo. In this work, we describe the amplification of representative cDNAs from morphologically and developmentally normal, individual human embryos at all stages from pronucleate to blastocyst. These cDNAs were probed to reveal the temporal expression pattern of cell adhesion molecules thought to play a key role in murine preimplantation embryo development. We demonstrated constitutive expression of beta actin, beta 1 and alpha 6 integrins, ZO-1 and E-cadherin, as shown previously in mouse embryos. No expression of beta 3, alpha 2, alpha 3 or alpha 7 integrins nor of L or P selectin was detected at any stage of preimplantation development. beta 5 integrin showed a regulated pattern of expression and was not expressed in blastocysts, while desmocollin-2 could only be detected at the blastocyst stage. Expression and localization of beta 1, beta 5 and alpha 6 integrins and ZO-1 and E-cadherin proteins was confirmed in blastocyst stage embryos by immunocytochemistry. We have identified differences in the expression of integrin molecules between mouse and human embryos, and propose a role for alpha v beta 5 and alpha 6 beta 1 integrin dimers in the human embryo at implantation.     

Prostacyclin receptor signaling and early embryo development in the mouse

BACKGROUND: Prostacyclin (PGI(2)) plays an important role in mouse embryo development and implantation. However, it is unclear whether its action is mediated via the I prostaglandin receptor (IP). METHODS: We compared the preimplantation development of IP deleted (IP-/-) embryos and wild-type (WT) embryos. We also evaluated the effect of iloprost, a stable PGI(2) analog, and L-165041, a peroxisome proliferator activated receptor delta (PPARdelta) ligand, on IP-/- versus WT embryos. Finally, we compared the development of heterozygous IP deficient embryos carrying a normal maternal IP allele versus paternal IP allele. RESULTS: Development of IP-/- embryos lagged behind WT embryos and was not enhanced by either the PGI(2) analog or the PPARdelta ligand. WT embryos had slightly higher, although statistically not significant, implantation rates than IP-/- embryos. Heterozygous IP deficient embryos carrying a normal maternal IP allele showed better development and responded to the PGI(2) analog, unlike those carrying the normal paternal IP allele. CONCLUSIONS: IP receptors play an important role in preimplantation embryo development and mediate the embryo's response to exogenous PGI(2). Early embryo development depends on the oocyte IP receptor.     

Expression of transforming growth factor-alpha and its receptor during human liver development and maturation

We investigated the expression of transforming growth factor-alpha (TGF-) and its receptor during human liver development and maturation, using immunohistochemistry. In the fetal liver, strong immunoreactivity for TGF- and its receptor was noted in intrahepatic bile duct cells of various developmental stages; moderate immunoreactivity for TGF- and mild immunoreactivity for TGF- receptor were found in immature hepatocytes. In the postnatal liver, reactivity for TGF- in hepatocytes decreased gradually and was negative or only weakly positive in the adult liver, while reactivity for TGF- receptor in hepatocytes increased gradually and was strongly positive in the adult liver. In contrast, immunoreactivity of TGF- and its receptor in intrahepatic bile duct cells persisted in the postnatal liver and was positive in the adult liver. These data suggest that the system of TGF- and its receptor has an important role in the proliferation and differentiation of intrahepatic biliary cells and hepatocytes in the fetal liver. The decreasing expression of TGF- in hepatocytes in the postnatal liver may indicate that proliferative activity of hepatocytes gradually decreases with liver maturation. The presence of TGF- and its receptor in intrahepatic bile ducts in the postnatal liver suggests that the system of TGF- and TGF- receptor is operative postnatally.     

Expression of glucose transporter isoforms and the insulin receptor during hamster preimplantation embryo development

During preimplantation development, embryos of many species are known to express up to five isoforms of the facilitative glucose transporter proteins (GLUT). Development of hamster blastocysts is inhibited by glucose. We therefore investigated GLUT isoform and insulin receptor (IR) expression in hamster preimplantation embryos cultured in glucose-free medium from the 8-cell stage onwards. We show that GLUT1, 3 and 8 mRNA are constitutively expressed from the 8-cell to the blastocyst stage. The IR is expressed from the morula stage onwards. Messenger RNA of the insulin-responsive GLUT4 was not detected at any stage. GLUT1 and 3 were localised by immunocytochemistry. GLUT1 was expressed in both embryoblast and trophoblast, in the latter, mainly in basal and lateral membranes directed towards the blastocoel and embryoblast. GLUT3 was exclusively localised in the apical membrane of trophoblast cells. We show that hamster preimplantation embryos express several GLUT isoforms thus closely resembling embryos of other mammalian species. Despite endogenous IR expression, the insulin-sensitive isoform GLUT4 was not expressed, indicating that the insulin-mediated glucose uptake known from classical insulin target cells may not be relevant for hamster blastocysts.