碱性成纤维细胞生长因子对骨髓基质细胞的生物学效应

目的探讨碱性成纤维细胞生长因子(bFGF)对骨髓基质细胞(BMSC)粘附、增殖和分化等生物学效应的影响.方法利用体视学计数、MTT法及ALP试剂盒分别测定不同浓度bFGF诱导一定时间后BMSG的粘附特性、增殖和分化情况的变化.结果10n/mLbFGF明显促进BMSG的粘附,但是200ng/mLbFGF反而不利于细胞粘附;在细胞增殖和分化测定中,100ng/mLbFGF明显促进细胞增殖,但细胞碱性磷酸酶含量也最低.结论碱性成纤维细胞生长因子对骨髓基质细胞的生物效应是复杂和多方面的,可以作用于骨髓基质细胞的粘附、增殖和分化等多个环节,这种影响与碱性成纤维细胞生长因子的剂量有关.     

Basic fibroblast growth factor fused to a signal peptide transforms cells

Basic fibroblast growth factor (bFGF) is a potent growth and angiogenic factor that is found in abundance in tissues such as brain, hypothalamus, kidney and cartilage. Despite this copious production of bFGF, most of these tissues are not undergoing either active growth or angiogenesis, suggesting that bFGF activity must be regulated so as to prevent autostimulation of cell growth. In cultured cells, bFGF is associated mainly with cells and basement membranes and is not released into the medium. Prevention of release could be a mechanism for regulation of bFGF activity and may be a consequence of the apparent absence of a secretory-signal sequence in the bFGF protein. Here we investigate whether this regulation can be overridden through the forced secretion of bFGF. Such secretion might provide the bFGF access to its receptor and in turn lead to autocrine transformation of the cell. We report that bFGF, as specified by a recombinant plasmid, is itself unable to induce such transformation, but acquires this ability after fusion with a secretory-signal sequence. The resulting transformants undergo unusual morphological alteration and display tumorigenicity.     

PDGF induction of tyrosine phosphorylation of GTPase activating protein

The cascade of biochemical events triggered by growth factors and their receptors is central to understanding normal cell-growth regulation and its subversion in cancer. Ras proteins (p21ras) have been implicated in signal transduction pathways used by several growth factors, including platelet-derived growth factor (PDGF). These guanine nucleotide-binding Ras proteins specifically interact with a cellular GTPase-activating protein (GAP). Here we report that in intact quiescent fibroblasts, both AA and BB homodimers of PDGF rapidly induce tyrosine phosphorylation of GAP under conditions in which insulin and basic fibroblast growth factor (bFGF) are ineffective. Although GAP is located predominantly in the cytosol, most tyrosine-phosphorylated GAP is associated with the cell membrane, the site of p21ras biological activity. These results provide a direct biochemical link between activated PDGF-receptor tyrosine kinases and the p21ras-GAP mitogenic signalling system.     

Capillary endothelial cells express basic fibroblast growth factor, a mitogen that promotes their own growth

Angiogenesis, the formation of new capillaries, which is observed in embryonic and injured tissue and is particularly prominent in the vicinity of solid tumours, involves the migration and proliferation of capillary endothelial cells. It is probably triggered by agents, such as basic fibroblast growth factor (bFGF), thought to be released from tissues adjacent to proliferating capillaries. As well as being a potent inducer of cell division in capillary endothelial cells in vitro, bFGF can act as an angiogenic agent in vivo. It is present in a wide variety of richly vascularized tissues including brain, pituitary, retina, adrenal gland, kidney, corpus luteum, placenta and various tumours. So far, however, the normal bFGF-producing cell species in these tissues have not been identified. We report here that capillary endothelial cells express the bFGF gene, that they produce and release bFGF and that bFGF derived from them can stimulate the proliferation of capillary endothelial cells. We conclude that bFGF can act as a self-stimulating growth factor for capillary endothelial cells, and that it is possible that the formation of new capillaries is induced by capillary endothelial cells themselves.     

Vaccinia virus encodes a polypeptide homologous to epidermal growth factor and transforming growth factor

Epidermal growth factor (EGF) and transforming growth factor type I (TGF) are polypeptides of 53 and 50 amino acid residues, respectively. Both bind to EGF receptor, a 1,200-residue transmembranous glycoprotein, leading to phosphorylation of the receptor, enhancement of its tyrosine-specific kinase activity and ultimately to stimulation of cell growth. We report here that a 140-residue polypeptide encoded by one of the early genes of vaccinia virus (VV) is related closely to EGF and TGF. The presence of putative signal and transmembranous sequences further suggests that the viral protein might be an integral membrane protein, but that, as in the case of EGF itself, the membrane-associated form may be the precursor of a soluble growth factor. Production of EGF-like growth factors by virally infected cells could account for the proliferative diseases associated with members of the poxvirus family such as Shope fibroma virus, Yaba tumour virus, and molluscum contagiosum virus (MCV).     

Photoreceptor degeneration in inherited retinal dystrophy delayed by basic fibroblast growth factor

Numerous inherited retinal degenerations exist in animals and humans, in which photoreceptors inexplicably degenerate and disappear. In RCS rats with inherited retinal dystrophy, the mutant gene is expressed in the retinal pigment epithelial (RPE) cell, and leads to the loss of photoreceptor cells. Photoreceptors can be rescued from degeneration if they are juxtaposed to wild-type RPE cells in experimental chimaeras or by the transplantation of RPE cells from normal rats. In both cases, the rescue effect extends beyond the immediate boundaries of the normal RPE cells, suggesting trophic action of a diffusible factor(s) from the normal RPE cells. We considered that the fibroblast growth factors, aFGF and bFGF, might have such a trophic role as they are found in the retina and RPE cells; bFGF acts as a neurotrophic agent after axonal injury in several regions of the central nervous system, and bFGF induces retinal regeneration from developing RPE cells. Here we report that subretinal injection of bFGF results in extensive rescue of photoreceptors in RCS rats for at least two months after the injection, and that intravitreal injection of bFGF results in even more widespread rescue, across almost the entire retina. The findings demonstrate for the first time that bFGF can act as a survival-promoting neurotrophic factor in a hereditary neuronal degeneration of the central nervous system.     

Long-term proliferation of mouse primordial germ cells in culture.

Primordial germ cells (PGCs) are first identifiable as a population of about eight alkaline phosphatase-positive cells in the 7.0 days postcoitum mouse embryo. During the next 6 days of development they proliferate to give rise to the 25,000 cells that will establish the meiotic population. Steel factor is required for PGC survival both in vivo and in vitro and together with leukaemia inhibitory factor stimulates PGC proliferation in vitro. In feeder-dependent culture, PGCs will proliferate for up to 7 days, but their numbers eventually decline and their proliferative capacity is only a fraction of that seen in vivo. Here we report a further factor that stimulates PGC proliferation in vitro, basic fibroblast growth factor (bFGF). Furthermore, bFGF, in the presence of steel factor and leukaemia inhibitory factor, stimulates long-term proliferation of PGCs, leading to the derivation of large colonies of cells. These embryonic germ cells resemble embryonic stem cells, pluripotent cells derived from preimplantation embryos, or feeder-dependent embryonal carcinoma cells, pluripotent stem cells of PGC-derived tumours (teratomas and teratocarcinomas). To our knowledge, these results provide the first system for long-term culture of PGCs.     

大鼠胎脑神经干细胞的分离和培养

胚龄14．5～16．5d(E14．5～16．5)的大鼠胎脑组织获得大鼠胎脑神经干细胞(rat fetal neural stem cells，rFNSCs)，培养于含有碱性成纤维细胞生长因子(basic fibroblast growth factor，bFGF)和表皮生长因子(epidermal growth factor，EGF)的无血清培养液DMEM／F12中，用^3[H]胸苷掺入试验检测EGF和bFGF对大鼠胚胎神经干细胞分裂和增殖的影响．BrdU结合反应和nestin免疫组化检测显示培养细胞在早期时代约90％以上具有分裂增殖能力并显示nestln阳性，而且这些细胞在培养过程中可以分化神经元、星形胶质细胞和少突胶质细胞，证明分离培养的是神经干细胞，可用于移植、定向分化和基因转移的研究．     

氨基酸定点突变碱性成纤维生长因子的稳定性

为了提高碱性成纤维生长因子的体外稳定性,分析比较了野生型人碱性成纤维细胞生长因子（hbFGF）和半胱氨酸（Cys）定点突变型hbFGF的生物活性、肝素结合能力、体外聚合程度和聚合成分,以及两种蛋白单体的表面静电分布和溶剂可及表面面积．结果表明,突变型hbFGF保持了野生型hbFGF的生物活性以及肝素结合能力,并显著了降低了体外聚合程度,而单体三维结构中的静电分布和溶荆可及表面面积变化不明显,由此可见,Cys突变影响了hbFGF的共价聚合,而对非共价聚合影响不大．     

Major reorganization of immunoglobulin VH segmental elements during vertebrate evolution

In mammals, the immunoglobulin heavy-chain variable region (VH) locus is organized in a linear fashion; individual VH, diversity (DH), joining (JH) and constant (CH) region segments are linked in separate regions. During somatic development, coding segments flanked by characteristic short recombination signal sequences, separated by intervening sequence regions that may exceed 2,000 kilobases (kb), are recombined. Combinatorial joining of different segments as well as imprecision in this process contribute to the diversity of the primary antibody response; subsequent mutation further alters functionally rearranged genes. This basic somatic reorganization mechanism is shared by six major families of genes encoding antigen receptors. Previously, we have shown that multiple germline genes and mammalian-like recombination signal sequences are associated with the VH gene family of Heterodontus francisci (horned shark), a primitive elasmobranch. Studies presented here demonstrate that segmental reorganization involving mammalian-like DH and JH segments occurs in the lymphoid tissues of this species. In marked contrast to the mammalian system, we find multiple instances of close linkage (approximately 10 kb) between individual VH, DH, JH, and CH segments. This unique organization may limit combinatorial joining and be a factor in the restricted antibody response of this lower vertebrate.     

Surface plasmon resonance analysis to evaluate the importance of heparin sulfate groups＇ binding with human aFGF and bFGF

Human acidic and basic fibroblast growth factors (aFGF and bFGF) are classic and well characterized members of the heparin-binding growth factor family. Heparin is generally thought to play an extremely important role in regulating aFGF and bFGF bioactivities through its strong binding with them. In order to unravel the mechanism of the interactions between heparin and FGFs, and evaluate the importance of heparin sulfate groups‘ binding with FGFs, surface plasmon resonance analyses were performed using IAsys Cuvettes System. Heparin and its regioselectively desulfated derivatives were immobilized on the cuvettes, aFGF and bFGF solutions with different concentrations were pipetted into the cuvettes and the progress of the interaction was monitored in real-time by Windows-based software, yielding kinetic and equilibrium constants for these interactions. In addition, in order to reduce the delicate difference among the cuvettes, inhibition analyses of mixture of FGFs and immobilized native heparin by modified heparins were also done. The data from these two methods were similar, indicating that all sulfate groups at 2-0, 6-0 and N- in heparin were required for the binding to aFGF; and that their contribution to the binding was in the order 2-O, N- and 6-O-sulfate group.In contrast, definite contribution of the 6-O-sulfate group to the binding with bFGF was most apparent, while the other two sulfate groups appeared to be necessary in the order 2-O and N-sulfate group. These methods established here can be used for analysing the effect of sulfate groups in heparin on the binding with other human FGF members or other heparin-binding proteins.     

重组人碱性成纤维细胞生长因子诱导Balb/c3T3细胞增殖的实验研究

目的研究不同浓度重组人碱性成纤维细胞因子对Balb/c 3T3细胞增殖活性的影响.方法重组质粒pGEX-bFGF转入到DH5α大肠杆菌体内,增菌培养表达bFGF,亲和层析纯化,制备8种不同药物浓度的bFGF,分别进行诱导Balb/c 3T3细胞增殖实验研究,MTT法检测各组细胞增殖活性.结果 bFGF诱导Balb/c 3T3细胞增殖存在浓度依赖性,最适浓度为4～16μg/L.结论 bFGF具有较好的诱导Balb/c 3T3细胞增殖的能力,为后续研究工作奠定实验基础.     

乳铁蛋白对口腔癌细胞中VEGF和bFGF表达的影响

目的探讨乳铁蛋白对口腔癌细胞中血管内皮生长因子(VEGF)和碱性成纤维细胞生长因子(b FGF)表达的影响.方法选用人口腔鳞癌细胞系CAL-27,分为0.006,0.013,0.025,0.050 g/L重组人乳铁蛋白实验组和空白对照组.应用RT-PCR法检测VEGF和b FGF mRNA表达水平;应用Western Blot法检测VEGF和b FGF蛋白表达水平.结果 CAL-27口腔癌细胞VEGF和b FGF mRNA均随着乳铁蛋白浓度增加表达量逐渐减少;0.050 g/L乳铁蛋白实验组VEGF和b FGF mRNA表达量明显低于空白对照组,差异具有统计学意义(P0.05).CAL-27口腔癌细胞VEGF和b FGF蛋白产物随着乳铁蛋白浓度增加表达量逐渐减少;0.050 g/L乳铁蛋白实验组VEGF,b FGF蛋白表达量明显低于空白对照组,差异具有统计学意义(P0.05).结论乳铁蛋白可有效抑制口腔癌细胞系CAL-27细胞中VEGF,b FGF的转录和表达,进而抑制血管生成,可作为乳铁蛋白抗口腔癌的机制之一.     

bFGF调控CD34对子宫内膜癌细胞生长的影响

目的探讨碱性成纤维细胞生长因子（bFGF）调控CD34对子宫内膜癌细胞生长的影响。方法免疫组化法检测正常子宫内膜和子宫内膜癌组织中bFGF、微血管密度（MVD）和增殖细胞核抗原（PCNA）的表达。用TUNEL原位凋亡法检测细胞凋亡并计算凋亡率（AR）。结果子宫内膜癌组织中bFGF、MVD和PCNA表达均明显高于正常子宫内膜组织,而AR低于正常子宫内膜组织。bFGF、MVD和PCNA均呈正相关。bFGF和MVD与AR均呈负相关。结论bFGF促进肿瘤细胞增殖,抑制肿瘤细胞凋亡。     

重组BbFGF工程菌的发酵条件

利用大肠杆菌BL21（DE3）pLysS生产重组牛碱性成纤维细胞生长因子(BbFGF),对此工程菌的发酵条件进行了研究，在前期试验的基础上，重点探索培养基、诱导剂及区葡萄糖添加方式对T7启动子指导下的bFGF合成的影响，根据摇瓶试验和分批发酵试验结果，建立了一套变速加葡萄糖的高密度补料分批发酵工艺，在此工艺下，经11h发酵，可得光密度为30.7、bFGF产量为95mg/L的发酵产物，两步法纯化目的蛋白，得到纯度为95%的重组bFGF，其生物活性和标准品一致。     

枳菊解郁汤对抑郁模型小鼠空间学习记忆及脑内bFGF的影响

目的：探讨枳菊解郁汤对抑郁模型小鼠空间学习记忆的影响及其机制。方法：采用多因素慢性不可预知应激源建立抑郁动物模型,并给予动物不同剂量枳菊解郁汤。通过Morris水迷宫实验,检测各组小鼠空间学习记忆能力的变化;采用免疫组织化学方法检测碱性成纤维细胞生长因子（bFGF）在脑内的表达。结果：抑郁模型组小鼠空间学习记忆能力明显下降（P〈0．05）;bFGF在前脑皮层和海马CA1,CA3及DG区的表达明显降低（P〈0．05）;与模型组小鼠相比,枳菊解郁汤中剂量组空间学习记忆能力明显增强（P〈0．05）;bFGF在海马的CA1、CA3和DG区及前脑皮层的表达明显增加（P〈0．05）。结论：枳菊解郁汤的抗抑郁作用可能与海马和前脑皮层bFGF表达的上调有关。     

新生大鼠海马神经干细胞的体外培养及鉴定

的探讨新生大鼠海马神经干细胞（NSC）的体外培养和诱导分化的条件和特点。方法分离出生1d大鼠海马,在表皮生长因子、碱性成纤维生长因子和B27联合作用下使其稳定增殖,用5-溴脱氧尿苷（BMU）标记处于增殖状态的神经干细胞,应用免疫荧光染色方法行巢蛋白（Nestin）、5-溴脱氧尿苷（BrdU）、β-Ⅲ型微管蛋白（Tuj-1）、波形蛋白（Vimenfin）和Galc-C免疫荧光染色,对NSC的增殖及其分化的细胞进行鉴定。结果体外培养的NSC增殖成神经干细胞球并传代,鉴定为Nestin染色阳性细胞和5-溴脱氧尿苷（BrdU）标记染色阳性细胞,并可诱导分化为神经元细胞（Tuj-1染色阳性细胞）、神经胶质细胞（Vimentin染色阳性细胞）和少突胶质细胞（GMc-C染色阳性细胞）。结论采用无血清培养基中加入特定生长因子的培养技术,可培养出在体外稳定增殖并有多向分化潜能的新生大鼠海马神经干细胞。     

基于Envisat数据的地物后向散射特征研究

本文采用ENVISAT ASAR的APMode（Alternating Polarisation Mode）数据,由雷达图像成像原理及物理机制人手分析广州市几种典型地物（水体、植被、建筑物）在不同极化方式下后向散射特征及其成因。在此基础上选取HH极化图像、VV极化图像及VH／VV比值图进行合成,并利用监督分类方法对该极化合成影像进行分类,与光学影像的分类结果图进行比较。结果表明：极化雷达具有探测水质的能力,建筑物的后向散射特征还与其排列方式有关,极化合成影像分类图能初步达到地物分类识别的效果。     

拉西地平和川芎嗪对大鼠缺氧性肺动脉高压及肺组织bFGF表达的影响

目的：观察拉西地平和川芎嗪预防大鼠常压缺氧性肺动脉高压（HPH）的疗效及探讨其对肺组织碱性成纤维细胞生工因子（bFGF)表达水平的影响,方法：应用常压缺氧方法建立HPH大鼠模型;采用右心导管法[测定肺动脉压;用免疫组化技术检测肺组织bFGF表达水平,结果：拉西地平和川芎嗪均能显著阻抑缺氧大鼠肺动脉压的升高,两药莪疗效无显著性差异,且肺动脉管壁增厚不明显,缺氧对照组大鼠肺内bFGF染色颗粒较正常对照组明显增加,而缺氧加拉西地平或川芎嗪组肺内bFGF染色颗粒较缺氧对照组明显减少,结论：拉西地平和川芎嗪均有预防HPH的作用,并可能通过抑制肺组织bFGF表达而调控肺血管结构重建。