Comparison of the expression patterns of several fibroblast growth factors during chick gastrulation and neurulation

The fibroblast growth factor family consists of a large number of secreted polypeptide growth factors. The developmental importance of the family has been highlighted in many studies, which serve to underscore their role as local instructive signals directing diverse processes in the developing embryo. We wished to characterize and compare the expression patterns of nine members of the fibroblast growth factor family in the chick embryo. In this study, we survey the expression patterns of fgf-2, -3, -4, -8, -10, -12, -13, -14 and -18 during gastrula and neurula stages (Hamburger and Hamilton stages: 4-13). As well as providing a comparison of the expression patterns of those fibroblast growth factors already published, we provide new data on the expression patterns of some of these genes at early stages.     

肠道缺血再灌流对肾内源性碱性成纤维细胞生长因子和 …

目的和方法：研究肠道缺血再灌流过程对肾脏内源性碱性成纤维细胞生长因子和转化生长因子β基因表达的影响，采用肠系膜上动脉夹闭４５ｍｉｎ与再灌流６ｈ和２４ｈ动物模型，用原位杂交与逆转录ＰＣＲ（ＲＴ－ＰＣＲ）技术研究两种生长因子基因在正常，缺血以及再灌流肾组织中的表达。     

An immunohistochemical study of basic fibroblast growth factor in the developing chick

An antiserum against basic fibrobrast growth factor (bFGF) was characterized by immunoblot experiments and used to investigate immunohistochemically the appearance of bFGF-like immunoreactivity in the developing chick. Crude homogenates of chick embryos at every developmental stage, when subjected to immunoblotting with the use of bFGF antiserum, exhibited a main band with the same molecular weight (18 kDa) as bovine bFGF. With immunohistochemistry, bFGF immunoreactivity (bFGF-IR) was detected exclusively in intracellular components of various tissues at different stages of development; bFGF-IR appeared initially on embryonic (incubation) day 3 (E3) in the myotome, on E12 in the spinal cord and ganglia, on E8 in chondrocytes and osteoblasts of the vertebrae, and on E10 in the esophageal epithelium. Immunoreaction products were present either in the cytoplasm or in the nuclei, depending on the types of individual bFGF-containing cells; developing chondrocytes and cells in the stratum basale of the esophagus exhibited intense immunoreactions exclusively within the nuclei, and the other cells mainly within the cytoplasm. Moreover, bFGF-IR was observed in discrete regions of these tissues at different stages; the epithelium of the esophagus containd bFGF-IR in all layers on E10 to E18 with a superficial-to-basal gradient, but it began to exhibit bFGF-IR only in the stratum basale after E20; and bFGF-IR was more abundant in hypertrophic chondrocytes than in proliferating ones. As chicks aged, bFGF-IR decreased or disappeared in the muscles, vertebrae and esophageal epithelium, but neuronal bFGF increased in intensity until the perinatal period and thereafter remained unchanged. These findings suggest that bFGF not only plays a pivotal role in regulating cell proliferation and differentiation in developing chick tissues, but also acts as a non-mitogenic mediator in nervous tissue.     

鼠成纤维细胞因子8的克隆和表达

目的 :克隆和表达鼠成纤维细胞因子 8(FGF 8) ,以便进一步研究FGF 8的功能。方法 :根据已发表的鼠成纤维细胞因子 8的cDNA序列 ,从鼠胚胎中提取总RNA ,采用RT PCR技术 ,用设计合成的引物钓取cDNA片段。将该cDNA片段定向插入酵母表达载体pYEX4T 1中 ,做DNA序列分析。用此重组质粒转化酿酒酵母 ,经Western印迹分析表达产物。用MTT和3H TdR掺入法检测重组蛋白活性。结果 :经序列分析证明 ,所钓取到的 6 0 0bp的cDNA片段确实是FGF 8的“b”选择性剪接型(FGF 8b)。Western印迹分析表明 ,该基因获得了较高效的表达 ,分子量约 5 5kD。实验组成纤维细胞株NIH3T3的MTT和3H TdR掺入量明显增多 ,拮抗组的掺入量有所下降。结论 :获得了有活性的重组鼠FGF 8蛋白 ,该蛋白能刺激NIH3T3生长 ,并且这种活性能被重组人FGFR 1的细胞外段所拮抗     

Expression of the type III TGFbeta receptor during chick organogenesis

Transforming growth factor beta (TGFbeta) is a regulator of embryonic development. The role of specific TGFbeta receptors is emerging, and a unique role for the type III TGFbeta receptor (TBRIII) has been suggested. We report the pattern of TBRIII expression in chicken embryos from 2 to 14 days in ovo.     

人成纤维细胞生长因子13基因在酵母细胞中的表达

目的：用酵母细胞表达重组人成纤维细胞生长因子（ｈＦＧＦ１３）蛋白并检测其活性。方法：通过ＲＴ－ＰＣＲ从流产胎儿脑组织中钓取ｈＦＧＦ１３ｃＤＮＡ,将其克隆入ｐＹＥＸ４Ｔ－１载体质粒。在酏母细胞表达ＧＳＴ＿ｈＦＧＦ１３融合蛋白。以细胞增殖实验测定酵母表达蛋白粗提物的活性。结果：ＧＳＴ－ｈＦＧＦ１３融合刷拓酵发母细胞中得到表达;ＧＳＴ－ｈＦＧＦ１３融合蛋白细胞ＮＩＨ３Ｔ３细胞的增殖,而且此活性能够被ＦＧ     

Changes of type I fibroblast growth factor receptor gene during development

目的研究人成纤维细胞生长因子受体1(FGFR1)基因在发育过程中的可能变化。方法采用Southernblot的方法对胎儿及多种组织基因组DNA进行分析。结果成人FGFR1基因与胚胎期的在基因组水平上是不同的。结论人FGFR1基因在发育过程中可能发生了重排或丢失,这种变化可能导致FGFR1在胚胎期和成年期的表达水平和分子结构的不同,从而改变细胞的功能状态。     

Basic fibroblast growth factor expression in isolated small human ovarian follicles

Basic fibroblast growth factor (bFGF) is involved in cell proliferation, differentiation, and angiogenesis. It has long been known that bFGF acts as a powerful mitogen for various mammalian granulosa cells in culture. To investigate the possible involvement of bFGF expression in follicle initiation and growth in vivo, we performed nested RT-PCR on ovarian cortical biopsies and quantitative PCR on human follicle populations isolated by laser capture microdissection. Using morphological criteria, follicles were characterized as putative non-growing, primary, or small secondary. RNA was extracted from samples, reverse-transcribed, and relative gene expression levels determined with TaqMan real-time PCR, using 18S rRNA as the endogenous control. Results confirmed bFGF expression in human adult ovarian cortex, and in the isolated follicles a down-regulation of bFGF mRNA was evident as small follicles develop. This study demonstrates a possible relationship between bFGF mRNA expression and follicle development.     

The role of maternally derived epidermal growth factor and the epidermal growth factor receptor during organogenesis in the rat embryo

Epidermal growth factor (EGF) has been implicated in the control of embryonic development, but although the receptor is expressed from an early stage, there is little evidence of embryonic expression of EGF. In order to investigate the role of maternally derived EGF during organogenesis, rat embryos were explanted at d 9.5 and cultured in serum depleted of low molecular weight molecules (retenate) which was then supplemented with EGF. Serum depleted of low molecular weight molecules by prolonged filtration loses its capacity to support normal embryonic development, possibly due to the loss of growth promoting factors. The addition of EGF to retenate significantly improved embryonic development with a maximal effect at 8 ng/ml. The addition of an analogue of EGF, long EGF, to retenate also caused a significant increase in development, although at higher concentrations a decrease in its effect was observed, possibly due to down regulation of the EGF receptor. Therefore, embryos may be able to utilise maternally derived EGF during organogenesis. To test the effects of inhibiting the EGF receptor during organogenesis, d 9.5 embryos were cultured in the presence of tyrphostin 47, a specific EGF receptor inhibitor. Tyrphostin 47 caused a significant dose-dependent decrease in the development of embryos which was also observed when tyrphostin 47 was injected into the vitelline circulation at d 11.5 to bypass the effects of the yolk sac. These findings suggest that the EGF receptor is essential for normal organogenesis and may play a role in the control of proliferation and differentiation. Although EGF is not expressed in the rat embryo at this stage, maternally derived EGF may be the ligand for the embryonic EGF receptor.     

以Bacmid-杆状病毒-昆虫细胞系统表达人FGF-9

目的：在Ｂａｃｍｉｄ杆状病毒昆虫细胞系统中表达人ＦＧＦ９ 。方法：采用ＲＴＰＣＲ技术,自新鲜人脑胶质瘤组织获取人ＦＧＦ９ 全编码区ｃＤＮＡ,将其克隆入ｐＣＲＴＭ Ⅱ质粒及ｐＹＥＸ４Ｔ１ 真核表达质粒,经ＤＮＡ自动测序仪进行ＤＮＡ序列测定。将人ＦＧＦ９ ｃＤＮＡ 定向克隆入ｐＦａｓｔＢａｃ 质粒,进一步将其转座入Ｂａｃｍｉｄ 中,在昆虫细胞Ｓｆ９ 中进行表达,采用ＳＤＳＰＡＧＥ对表达产物进行分析。结果：在昆虫细胞表达系统中表达出人ＦＧＦ９ 重组蛋白。结论：人ＦＧＦ９ 在Ｂａｃｍｉｄ杆状病毒昆虫细胞系统中得到了表达。     

Coordinate loss of fibroblast growth factor 2 and laminin 5 expression during neoplastic progression of mammary duct epithelium

Branching morphogenesis in mammary ducts is associated with the expression of a number of proteins. These include laminin 5 and basic fibroblast growth factor (FGF)-2. Both proteins are lost with malignant transformation of mammary epithelium and have causal roles in branching morphogenesis in breast cancer cells in vitro. The in vivo relationships of these proteins with each other and with the loss of branched structures and mammary ductal dedifferentiation are not known. We carried out indirect fluorescence staining on subsets of archived pathologic samples from 55 patients, with a total of 140 pathologic entities, many with multiple stages of dedifferentiation present on the same cut, using antibodies to fibroblast growth factor-2 (FGF-2), fibroblast growth factor receptor-1 (FGFR1), and laminin 5 to determine expression. We also used Western blots to detect laminin 5 expression in MCF-7, T-47D, and MDA-MB-231 cells transfected with vectors constitutively expressing FGF-2 and immunofluorescence staining of matrix proteins deposited by these cells to determine export and accumulation of laminin 5. FGF-2 and laminin 5 expression were found throughout benign and atypical dedifferentiation in mammary tissue samples and were lost primarily with transformation to invasive cancer. FGFR1 was expressed in all cell types. Cancer cells enforced to express FGF-2 did not have detectable laminin 5 on Western blot, but matrix proteins deposited in culture did stain positive, suggesting accumulation of exported laminin 5. Data suggest roles for FGF-2 and laminin 5 in ductal integrity during mammary carcinogenesis, with loss of expression corresponding to loss of ductal structure. In vitro data suggest FGF-2 as causal in laminin 5 expression and export. Down-regulation of FGF-2 during transformation may contribute to loss of laminin 5 expression.     

小鼠发育期卵巢成纤维细胞生长因子10的表达

目的 研究小鼠卵巢发育期成纤维细胞生长因子10(FGF10)的表达. 方法 分离孕E14.5、E16.5、E18.5、生后第1天和3周龄小鼠卵巢,每组实验动物6只,应用免疫组织化学技术研究FGF10蛋白在卵巢中的表达;3周龄小鼠腹腔注射孕马血清促性腺激素(PMSG)48h后,再注射人绒毛膜促性腺激素(hCG),hCG注射后3h提取总mRNA,应用RT-PCR技术研究卵巢中FGF10、黄体生成素受体(LHR)、成纤维细胞生长因子7(FGF7)和成纤维细胞生长因子受体2Ⅲb(FGFR2Ⅲb)基因的表达变化. 结果 FGF10蛋白表达于卵母细胞,并且FGF10在生后第1天小鼠的卵母细胞上表达最强;两种促性腺激素处理后,FGF10 mRNA 与对照组相比表达下降,LHR mRNA表达升高,FGF7和FGFR2Ⅲb mRNA表达不变. 结论 FGF10蛋白特异表达于小鼠卵母细胞膜,促性腺激素影响卵巢FGF10 mRNA的表达,推测FGF10可能参与调控小鼠卵母细胞的生长和卵泡发育.     

Changes in astrocytic basic fibroblast growth factor expression during and after prolonged exposure to escalating doses of amphetamine

We have shown that brief exposure to amphetamine leads to sustained glutamate-dependent increases in expression of the neurotrophic, neuroprotective factor, basic fibroblast growth factor, in astrocytes in dopaminergic cell body regions and that blockade of basic fibroblast growth factor in this region prevents the development of behavioral sensitization to amphetamine. Here we examine the effects of prolonged exposure to an escalating-dose regimen of amphetamine known to induce long-lasting sensitization to amphetamine and leading to increases in neuronal dendritic length and spine density in nucleus accumbens and prefrontal cortex and to decreases in spine density in occipital cortex. Astrocytic basic fibroblast growth factor immunoreactivity was increased in both dopaminergic cell body and terminal regions one week after termination of a two-week amphetamine treatment (1-4mg/kg). These effects were not evident one week after a five-week treatment (1-9mg/kg) and, in fact, one month later basic fibroblast growth factor levels in cell body regions were decreased. In the occipital cortex, basic fibroblast growth factor immunoreactivity was decreased one week after the two-week amphetamine treatment, but was not different from that seen in saline-treated animals after the five-week treatment. Increased astrocytic basic fibroblast growth factor expression appears to be an early, but relatively prolonged, response to amphetamine exposure and seems to parallel structural changes induced by repeated drug exposure.These findings suggest that basic fibroblast growth factor may participate in the development of structural changes brought about by amphetamine. The fact that the basic fibroblast growth factor response is not maintained after prolonged intense exposure to amphetamine suggests that the factors that initially induce basic fibroblast growth factor expression are self-regulating.     

斑马鱼胚胎发育过程成纤维细胞生长因子受体基因时序性表达

目的探讨斑马鱼胚胎发育过程成纤维细胞生长因子(FGF)受体基因时序性表达规律。方法分别提取发育至4、6、10、12、18、24、48及72hpf斑马鱼胚胎总RNA并反转录为cDNA,用实时定量PCR方法检测FGF受体基因(fgfrs)mRNA的相对表达量。结果斑马鱼胚胎发育过程fgfrs mRNA动态表达水平有明显峰谷变化(P0.01),胚胎fgfrs开始表达阶段依次为fgfr1a与fgfr1b(囊胚期)、fgfr4(原肠胚初期)、fgfr2(原肠胚期)及fgfr3(原肠胚后期),其表达高峰期分别在原肠胚期、原肠胚后期、体节期及胚胎发育中后期。旁系同源基因fgfr1a与fgfr1b动态表达趋势相似(r=0.830,P0.05)。fgfr1a与fgfr3、fgfr1b与fgfr4、fgfr2与fgfr3 mRNA表达量之间相关分析也有意义(r值分别为-0.726,0.821及0.772,P0.05)。结论斑马鱼胚胎发育过程fgfrs时序性表达有一定规律性,fgfr1、fgfr4主要参与胚胎早期发育调控,fgfr2、fgfr3主要参与胚胎中、后期发育调控。fgfr1a与fgfr1b共表达使其调控作用具有冗余能力。fgfr1和fgfr3之间在表达上可能存在相互制约关系。     

Vascular leakage in chick embryos after expression of a secreted binding protein for fibroblast growth factors

Fibroblast growth factors (FGFs) have been implicated in a variety of physiologic and pathologic processes from embryonic development to tumor growth and angiogenesis. FGFs are immobilized in the extracellular matrix of different tissues and require release from this storage site to trigger a response. Secreted FGF-binding proteins (FGF-BPs) can release immobilized FGFs, enhance the activity of locally stored FGFs and can thus serve as an angiogenic switch molecule in cancer. Here, we report on the effect of human FGF-BP transgene expression in chicken embryos. To establish the transgenic model, plasmid-based reporter vectors expressing luciferase, beta-galactosidase or green fluorescent protein were introduced through different routes into 4- to 5-day-old embryos grown outside their egg shell on top of the yolk sac. This allows for easy manipulation and continuous observation of phenotypic effects. Expression of human FGF-BP induced dose-dependent vascular permeability, hemorrhage and embryonic lethality. Light and electron microscopic studies indicate that this hemorrhage results from compromised microvascular structure. An FGF-1 expression vector with an added secretory signal mimicked this vascular leakiness phenotype whereas wild-type FGF-1 required coexpression of a threshold amount of FGF-BP. This model is a powerful tool for real-time monitoring of the effects of transient transgene expression during embryogenesis.     

力学刺激对角膜成纤维细胞碱性成纤维细胞生长因子表达的影响

目的研究在体及离体条件下不同力学环境对碱性成纤维细胞生长因子(basic fibroblast growth factor,bF-GF)表达的影响,探索力学因素在准分子激光原位角膜磨镶术(laser assisted in situ keratomileusis,LASIK)术后角膜损伤修复中的作用。方法建立不同切削量的LASIK手术动物模型,使在体角膜处于不同力学环境中,并于LASIK术后1周和1月处死实验动物提取组织蛋白。此外,采用Flexcell 4 000细胞力学加载系统对原代提取的兔角膜成纤维细胞施加频率为0.1 Hz、拉伸幅度分别为5%、10%和15%的周期性机械拉伸,并于拉伸后6 h和24 h后取细胞培养液上清。采用ELISA方法检测bFGF含量。结果 LASIK术后1周,角膜基质床残余30%组与对照组相比,bFGF含量显著增高(P<0.05);术后1月回落至正常水平,各组之间无显著性差异。不同时间点同一手术方式之间比较发现,仅30%组1周和1月有显著差异(P<0.05)。体外周期性拉伸实验表明拉伸6 h后1,5%拉伸组bFGF含量较对照组显著增高(P<0.05)2,4 h后显著性降低(P<0.05)。结论力学因素参与了早期角膜组织及角膜成纤维细胞bFGF表达的调节,bFGF在LASIK术后角膜组织修复中发挥了一定作用。