碱性成纤维细胞生长因子对生长板软骨细胞增殖与分化的影响

目的:探讨碱性成纤维细胞生长因子(bFGF)对生长板软骨细胞增殖和分化的作用。方法:分离并在低血清条件下培养兔生长板软骨细胞。采用改良MTT法检测细胞增殖倍数;羟脯氨酸法测定软骨细胞胶原产量;酶动力学方法测定碱性磷酸酶(ALP)活性。结果:bFGF浓度在5-100ng／ml范围内可以促进软骨细胞增殖,并以25ng／ml刺激时效果最为显著。当bFGF浓度高于25ng／ml时,抑制软骨细胞的胶原合成;当高于1ng／ml时,抑制碱性磷酸酶活性。结论:bFGF刺激生长板软骨细胞增殖,并在较高浓度时抑制生长板软骨细胞的分化。     

Inhibition of skeletal muscle satellite cell differentiation by transforming growth factor-beta

Skeletal muscle satellite cells were cultured from mature rats and were treated in vitro with transforming growth factor-beta (TGF-beta). Muscle-specific protein synthesis and satellite cell fusion were used as indicators of muscle differentiation; a dose-dependent inhibition of differentiation was observed in response to TGF-beta. In addition, TGF-beta depressed cell proliferation in a dose-dependent manner. Half-maximal inhibition of differentiation was seen with a TGF-beta concentration of approximately 0.1 ng/ml. Although proliferation was not inhibited, it was depressed and half-maximal suppression of proliferation occurred in response to 0.1-0.5 ng TGF-beta/ml. Neonatal rat myoblasts were also subjected to TGF-beta treatment, and similar results were observed. Neonatal cells, however, were more sensitive to TGF-beta than satellite cells, as indicated by the reduced concentrations of TGF-beta required to inhibit differentiation and reduce the rate of proliferation. Under identical culture conditions proliferation of muscle-derived fibroblasts were also depressed. The differentiation inhibiting effect of TGF-beta on satellite cells was reversible. It has been suggested that TGF-beta could be an important regulator of tissue repair, and its in vitro effects on satellite cells suggest a possible role in regulation of muscle regeneration.     

Basic fibroblast growth factor and transforming growth factor beta-1: Synergistic mediators of angiogenesis in vitro

We investigated the relative roles of basic fibroblast growth factor (bFGF) and transforming growth factor beta-1 (TGF-b) on bovine aortic endothelial cell mitogenesis and morphogenesis using two-dimensional Petri dish cultures and a threedimensional hydrated collagen gel. bFGF alone stimulated endothelial cell proliferation with an EC₅₀ of 0.5 ng/ml. At bFGF levels greater than 2.5 ng/ml, morphologic alterations in confluent monolayers predominated; cells changed from a cobblestone morphology to an elongated cell pattern and showed enhanced migration into a denuded area of a Petri dish. In the three-dimensional model, exposure of endothelial cell monolayers to high bFGF levels stimulated minor cell migration directly under the monolayer but no invasion into the gel matrix. In combination with bFGF, heparin potentiated morphogenic changes, but not mitogenesis. bFGF, modification of the antiproliferative effect of TGF-b in confluent cultures was evidenced by induction of endothelial cell sprouting in response to 0.5 ng/ml TGF-b and 10–20 ng/ml bFGF in two-dimensional cultures. On collagen gels, endothelial cells migrated into the deep layers of the gel in a dose-dependent manner: invasion was maximal at 0.3–0.7 ng/ml TGF-b with decreased invasion at higher concentrations. The optimal collagen concentration that supported cell invasion was 0.075% collagen with the number of invading cells decreasing with increasing collagen gel density. By scanning electron microscopy, invading endothelial cells assumed a fibroblast-like appearance with slender cell extensions. We concluded that bFGF and TGF-b had independent effects on endothelial cell morphology and mitogenesis in culture. In combination at specific doses, these agents stimulated sprouting in the two-dimensional model and cell invasion in a collagen gel model. Morphogenic changes may be the primary event in determining angiogenesis. © 1993 Wiley-Liss, Inc.     

Effects of epidermal growth factor and transforming growth factor-beta 1 on rat heart endothelial cell anchorage-dependent and -independent growth

This report describes the effects of epidermal growth factor (EGF) and transforming growth factor-beta 1 (TGF-beta 1) on the anchorage-dependent and -independent growth of rat heart endothelial cells (RHE-1A). When RHE-1A cells were grown in monolayer culture with medium containing 10% fetal bovine serum (FBS) supplemented with epidermal growth factor (0.1-100 ng/ml), growth was stimulated fivefold when compared to that of cells grown in medium containing 10% FBS alone. The stimulatory effect of EGF on RHE-1A cell monolayer growth was dose-dependent and half-maximal at 5 ng/ml. The addition of TGF-beta 1 in the range 0.1-10 ng/ml had no effect on RHE-1A cell monolayer growth when added to medium containing 10% FBS alone or 10% FBS supplemented with EGF (50 ng/ml). RHE-1A cells failed to grow under anchorage-independent conditions in 0.3% agar medium containing 10% FBS. In the presence of EGF, however, colony formation increased dramatically. The stimulatory effect of EGF was dose-dependent in the range 0.1-100 ng/ml and was half-maximal at 5 ng/ml. In contrast to its effects under anchorage-dependent conditions, TGF-beta 1 (0.1-10 ng/ml) antagonized the stimulatory effects of EGF on RHE-1A cell anchorage-independent growth. The inhibitory effect of TGF-beta 1 was dose-dependent and half-maximal at 0.1 ng/ml. EGF-induced RHE-1A soft agar colonies were isolated and reinitiated in monolayer culture. They retained the cobblestone morphology and contact-inhibition characteristic of normal vascular endothelial cells. Each of the clones continued to express Factor VIII antigen. These findings suggest that TGF-beta may influence not only endothelial cell proliferation but also anchorage dependence. These effects may in turn be of relevance to endothelial cell growth and angiogenesis in vivo.     

Recombinant human osteogenic protein-1 (hOP-1) induces new bone formation in vivo with a specific activity comparable with natural bovine osteogenic protein and stimulates osteoblast proliferation and differentiation in vitro.

We reported previously that a 32-36-kDa osteogenic protein purified from bovine bone matrix is composed of dimers of two members of the transforming growth factor (TGF)-beta superfamily: the bovine equivalent of human osteogenic protein-1 (OP-1) and bone morphogenetic protein-2a, BMP-2a (BMP-2). In the present study, we produced the recombinant human OP-1 (hOP-1) in mammalian cells as a processed mature disulfide-linked homodimer with an apparent molecular weight of 36,000. Examination of hOP-1 in the rat subcutaneous bone induction model demonstrated that hOP-1 was capable of inducing new bone formation with a specific activity comparable with that exhibited by highly purified bovine osteogenic protein preparations. The half-maximal bone-inducing activity of hOP-1 in combination with a rat collagen matrix preparation was 50-100 ng/25 mg of matrix as determined by the calcium content of day 12 implants. Evaluation of hOP-1 effects on cell growth and collagen synthesis in rat osteoblast-enriched bone cell cultures showed that both cell proliferation and collagen synthesis were stimulated in a dose-dependent manner and increased 3-fold in response to 40 ng of hOP-1/ml. Examination of the expression of markers characteristic of the osteoblast phenotype showed that hOP-1 specifically stimulated the induction of alkaline phosphatase (4-fold increase at 40 ng of hOP-1/ml), parathyroid hormone-mediated intracellular cAMP production (4-fold increase at 40 ng of hOP-1/ml), and osteocalcin synthesis (5-fold increase at 25 ng of hOP-1/ml). In long-term (11-17 day) cultures of osteoblasts in the presence of beta-glycerophosphate and L(+)-ascorbate, hOP-1 markedly increased the rate of mineralization as measured by the number of mineral nodules per well (20-fold increase at 20 ng of hOP-1/ml). Direct comparison of TGF-beta 1 and hOP-1 in these bone cell cultures indicated that, although both hOP-1 and TGF-beta 1 promoted cell proliferation and collagen synthesis, only hOP-1 was effective in specifically stimulating markers of the osteoblast phenotype.     

高糖和bFGF对骨髓间充质干细胞成骨分化的影响

目的：骨组织的形成是一个复杂的过程,受多种因素的影响,糖尿病所导致的持续高血糖对于成骨分化的影响机制尚不明确,以及在此分化过程中的各种细胞因子的作用机理仍不明了,现拟通过体外成骨诱导环境,观察高糖和碱性成纤维细胞生长因子（fibroblastgrowthfactorbFGF）对人骨髓间充质干细胞（humanmesenchymalstemcellshMSCs）成骨分化的影响。方法：hMSC在5．5mmol／L和25mmol／L葡萄糖浓度下培养6天,使用cck一8法测定各组细胞增殖情况;hMSC在两种糖浓度下成骨诱导28天,通过碱性磷酸酶（ALP）活性检测、茜素红染色、钙结节半定量检测,对比各组成骨分化活性;在两种糖浓度成骨诱导液中加入10ng／mlbFGF,使用RT—PCR技术检测各组细胞OCN、OPNmRNA表达差异。结果：高糖较正常糖浓度细胞增殖率下降,ALP活性降低,茜素红染色钙结节量减少,RT—PCR检测结果显示25mmol／L组OCN、OPNmRNA表达量低于5．5mmol／L组,加入bFGF后,25mmol／L组仍低于5．5mmol／L组,与未添加bFGF同葡萄糖组比较表达增加。结论：高糖使hMSC增殖能力下降,在成骨分化的过程中ALP活性降低,成骨相关基因OCN、OPN表达量下降,证明了高糖对hMSC成骨分化具有抑制作用,当加入bFGF后,改善了高糖对hMSC的抑制作用,提示糖尿病条件下高糖的存在是导致hMSC成骨分化能力下降的不利因素,同时初步证明了bFGF参与了成骨分化的过程,从而为在分子水平探讨糖尿病患者种植义齿骨结合形成相关机制奠定初步的基础．．     

细胞因子对鸡胚胎原始生殖细胞（EPGCs）增殖的影响

采用MTT法分别检测mLIF、bFGF、hSCF、hIL-11四种细胞因子协同作用对体外培养的第19、28期鸡EPGCs生长的影响。结果表明：与对照组相比较,19期的EPGCs体外培养72h后, mLIF、hSCF、bFGF、hIL-11对鸡EPGCs的增殖影响显著（P<0.05）。mLIF的最佳作用剂量是10~20ng/ml,hSCF的最佳作用剂量是15~20ng/ml,bFGF的最佳作用剂量是10～20ng/ml。hSCF、bFGF的联合使用优于单因子作用的结果（P<0.05）。单独使用hIL-11时,细胞的增殖情况比其他三因子单独使用的效果较差,但OD均值有随剂量增高而上升的趋势。在与其他因子联合使用的情况下, hIL-11的最佳作用剂量为0.10~0.20ng/ml。     

Basic fibroblast growth factor and insulin-like growth factor I are strong mitogens for cultured mouse keratinocytes

Basic fibroblast growth factor (bFGF), but not acidic fibroblast growth factor (aFGF), was found to be mitogenic for cultured mouse keratinocytes. A six-to-nine fold increase in 3H-thymidine (3H-dT) incorporation into the acid insoluble pool and a similar increase of the labeling index can be measured when bFGF, at a concentration between 1 and 10 ng/ml, is added to keratinocytes arrested in serum-free and growth factor-free medium with a Ca++-concentration below 0.1 mM. The half-maximal response is observed between 0.2 and 0.7 ng/ml. In the same culture system, insulin-like growth factor I/somatomedin C (IGF-I) and insulin act as mitogens. IGF-I shows half-maximal stimulation with 2-3 ng/ml, insulin with 100-500 ng/ml. Basic FGF, IGF-I and insulin can be classified as strong stimulators of DNA synthesis in mouse keratinocytes. In this regard they are comparable to epidermal growth factor, which shows a half-maximal stimulation at a concentration between 1.5-2 ng/ml. These results show that in addition to mesenchymal cells, FGF is a growth factor not only for neuroectodermal cells, but ectodermal cells in general. They further support the idea that the growth promoting effect of insulin on keratinocytes may be mediated by the IGF-I receptor.     

FSH and growth factors affect the growth and endocrine function in vitro of granulosa cells of bovine preantral follicles

The hypothesis was tested that bovine preantral follicles can be stimulated to grow in vitro by FSH and by the mitogens, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), but not by transforming growth factor-beta (TGFbeta), which generally inhibits EGF and bFGF action. Preantral follicles, 60 to 179 mum in diameter, were isolated from fetal ovaries by treatment with collagenase and DNase and cultured for 6 d in serum-free medium, with or without FSH and growth factors. Basic FGF (50 ng/ml), and to a lesser extent FSH (100 ng/ml) and EGF (50 ng/ml), stimulated thymidine incorporation by granulosa cells in bovine preantral follicles compared to control cultures (8-, 4- and 2.5-fold the labeling index of the controls; P < 0.05). Alone TGFbeta (10 ng/ml) had no effect on (3)H-thymidine incorporation, but it completely inhibited the bFGF- but not the FSH-stimulated increase in the labeling index and mean follicular diameter of preantral follicles (P < 0.05). By the end of the culture period oocytes in most treatments had degenerated, and the few surviving oocytes were in preantral follicles cultured with FSH or bFGF. Progesterone accumulation was greater (P < 0.05) in the presence of FSH (100 ng/ml) or EGF (50 ng/ml) than with bFGF, TGFbeta or control medium. Basic FGF strongly inhibited the effect of FSH on progesterone secretion (P < 0.05). Only FSH stimulated the conversion of exogenous testosterone to estradiol and both bFGF and TGFbeta markedly inhibited FSH-stimulated estradiol accumulation. These results indicate that proliferation of granulosa cells of bovine preantral follicles can be stimulated by bFGF, FSH and EGF, whereas TGFbeta inhibits growth, and that they are steroidogenically active in culture. Basic FGF and TGFbeta antagonize FSH-stimulated steroid production by granulosa cells of cultured bovine preantral follicles.     

生长因子TGF-β1和bFGF促兔骨髓间质干细胞向韧带样细胞分化的实验研究

摘要目的：采用生长因子TGF-β1和bFGF诱导体外培养的兔骨髓间质干细胞（MSCs）,转化为韧带样细胞,并研究此种韧带样细胞的生物特性。方法：自幼兔四肢骨抽取骨髓分离纯化MSCs并培养、增殖;采用特定浓度TGF-β1（10ng／ml）和bFGF（25ng／mL）对MSCs进行诱导分化,观察生长因子对MSCs生长、形态的影响,使用MTT法绘制细胞生长曲线,使用天狼腥红染色法定量对比MSCs分泌胶原蛋白量。单纯培养和单一因子诱导组作为对照。结果：TGF-β1和bFGF联合使用组,细胞形态优于空白组及单一因子组,细胞增殖率、胶原分泌量也均高于对照组。结论：联合使用生长因子TGF-β1和bFGF刺激兔MSCs,能够促使兔MSCs定向转化为韧带样细胞,对组织工程前交叉韧带的构建具有积极意义。     

C6 glioma cells produce basic fibroblast growth factor that can stimulate their own proliferation

With purified preparations of basic fibroblast growth factor (bFGF), we studied the effect of its growth-promoting activity on C6 glioma cells. We also examined with its antibody whether the cultured glioma cells could produce it. It was shown that bFGF stimulated the DNA synthesis and proliferation of C6 glioma cells in serum-free medium, and that the activity was potentiated by heparin, the bFGF concentrations for half-maximal stimulation being 0.2 and 5 ng/ml in the presence and absence of heparin, respectively. This effect of heparin was dose-dependent and was half-maximal at 0.5 microgram/ml. Next, we raised the antiserum against bFGF and detected a single immunoreactive band from extracts of C6 glioma cells by immunoblot analysis. The immunoreactive substance was partially purified on a heparin-Sepharose column and was shown to stimulate the DNA synthesis of C6 glioma cells. On the basis of its immunoreactivity, molecular weight, affinity for heparin, and growth-promoting activity, this substance was identified as bFGF. The content of bFGF in the cells was elevated as the cell density increased, but no immunoreactivity was detected in the conditioned medium of the cells. These results suggest that C6 glioma cells produce and store bFGF which is potent in stimulating their own growth.     

Basic Fibroblast Growth Factor Stimulates the Proliferation of Bone Marrow Mesenchymal Stem Cells in Giant Panda (Ailuropoda melanoleuca)

It has been widely known that the giant panda (Ailuropoda melanoleuca) is one of the most endangered species in the world. An optimized platform for maintaining the proliferation of giant panda mesenchymal stem cells (MSCs) is very necessary for current giant panda protection strategies. Basic fibroblast growth factor (bFGF), a member of the FGF family, is widely considered as a growth factor and differentiation inducer within the stem cell research field. However, the role of bFGF on promoting the proliferation of MSCs derived from giant panda bone marrow (BM) has not been reported. In this study, we aimed to investigate the role of bFGF on the proliferation of BM-MSCs derived from giant panda. MSCs were cultured for cell proliferation analysis at 24, 48 and 72 hrs following the addition of bFGF. With increasing concentrations of bFGF, cell numbers gradually increased. This was further demonstrated by performing 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) cell proliferation assay, 5-Bromo-2-deoxyUridine (BrdU) labeling and cell cycle testing. Furthermore, the percentage of MSCs that were OCT4 positive increased slightly following treatment with 5 ng/ml bFGF. Moreover, we demonstrated that the extracellular signal-regulated kinase (ERK) signaling pathway may play an important role in the proliferation of panda MSCs stimulated by bFGF. In conclusion, this study suggests that giant panda BM-MSCs have a high proliferative capacity with the addition of 5 ng/ml bFGF in vitro.     

Amylase secretion from saponin-permeabilized parotid cells evoked by cyclic AMP

Adenosine 3',5'-monophosphate (cAMP) evoked amylase release from saponin-permeabilized parotid cells of the rat. Saponin concentration was optimal at 10 micrograms/ml. Amylase release was stimulated by cAMP almost as well in Ca2+-free medium containing 1 mM EGTA as in the medium containing a physiological concentration of calcium. Although the basal and stimulated releases of amylase were markedly reduced by the further addition of 5 mM EGTA, the effect of cAMP was still detectable. The half-maximal dose of cAMP was 0.3 mM, whereas those of dibutyryl cAMP and 8-bromo-cAMP were 10-fold lower than that of cAMP. In the presence of 10 microM 3-isobutyl-1-methylxanthine, the half-maximal dose of cAMP was also decreased by 5-fold. These results suggest: 1) intracellular calcium is not essential for the exocytosis of amylase stimulated by cAMP; 2) the responsiveness of the cells to exogenous cAMP is reduced by phosphodiesterase.     

内皮素和bFGF对MC的促增殖作用及胰岛素的协同效应

采用３Ｈ－ＴｄＲ参入法,测定碱性成纤维细胞生长因子（ｂＦＧＦ）、胰岛素和内皮素－１（ＥＴ－１）对体外培养的大鼠肾小球系膜细胞（ＭＣ）增殖的影响,以及胰岛素与ｂＦＧＦ或ＥＴ－１促ＭＣ增殖的协同作用。结果表明,不同浓度的ｂＦＧＦ（５－２００ｎｇ／ｍｌ）和胰岛素（０．１－２．４Ｕ／ｍｌ）均显著升高ＭＣ的３Ｈ－ＴｄＲ参入值（ｃｐｍ值）（Ｐ＜０．０１）。ＥＴ－１对ＭＣ的ｃｐｍ值的影响依剂量不同呈现两种不同的效应,在１０－９－１０－７ｍｏｌ／Ｌ时,随着浓度的升高,ＭＣ的ｃｐｍ值明显升高（Ｐ＜０．０１）,并以１０－８ｍｏｌ／Ｌ作用最强;当升高到１０－６ｍｏｌ／Ｌ时,ＭＣ的ｃｐｍ值出现降低趋势。胰岛素与ｂＦＧＦ或低浓度ＥＴ－１（≤１０－８ｍｏｌ／Ｌ）共同作用于ＭＣ时,ＭＣ的ｃｐｍ值明显高于二者单独作用之和（Ｐ＜０．０１）,与高浓度ＥＴ－１（＞１０－７ｍｏｌ／Ｌ）共同作用于ＭＣ时,ＭＣ的ｃｐｍ值小于二者单独作用之和（Ｐ＞０．０５）。上述结果说明,胰岛素、ｂＦＧＦ和ＥＴ－１均能显著促进ＭＣ增殖;胰岛素与ｂＦＧＦ或低浓度的ＥＴ－１促ＭＣ增殖具有正协同作用,与高浓度ＥＴ－１呈现负协同作用。     

Epidermal growth factor promotes the chemotactic migration of cultured rat intestinal epithelial cells

The RIE-1 cell line is an untransformed, epithelial cell line derived from the rat small intestine. We report that epidermal growth factor (EGF), which regulates the proliferation of RIE-1 cells, also directs their movement. We measured cell migration through gelatin-coated filters in blind-well Boyden chambers. The migration of RIE-1 cells was stimulated up to approximately 100-fold by EGF, with a half-maximal response at 1-2 ng/ml and a maximal effect at 10 ng/ml. Further analysis showed that the RIE-1 cells responded directionally to a gradient of EGF in solution. Other growth factors tested did not stimulate RIE-1 cell migration, and EGF did not stimulate the migration of fibroblasts in this assay. We conclude that EGF is a potent and specific chemo-attractant for RIE-1 intestinal epithelial cells and suggest that EGF might influence epithelial cell migration in vivo.     

Induction of circulating neonatal stem cell populations

Hematopoietic cell differentiation and growth are regulated by paracrine molecules that include insulin and insulin-like growth factors (IGFs). IGF-I and -II stimulation of erythropoiesis in cultures of adult bone marrow and peripheral blood cells and murine fetal liver cells has been previously reported. In order to investigate whether these paracrines also influence differentiation and proliferation of human neonatal progenitor cells, we assessed their effects in cultures of umbilical cord blood and adult blood and marrow cells, using a serum-substituted system. IGF-I stimulated colony-forming unit-erythroid (CFU-E)-derived colony formation by adult cells by up to 265%, while IGF-II augmented colony formation by up to 100% in the presence of erythropoietin. Stimulation occurred in a saturable fashion over concentrations of 0 to 200 ng/ml. Similar results were obtained in subcultures of adult-circulating progenitors. Moreover, a subpopulation of erythropoietin-independent adult CFU-E was stimulated to proliferate by IGF-I but not by IGF-II. In contrast to these effects in adult marrow culture, IGF-II exerted a greater stimulatory effect on neonatal CFU-E proliferation than did IGF-I in erythropoietin-containing cultures. Additionally, IGF-II stimulated proliferation of erythropoietin-independent neonatal CFU-Es in a concentration-dependent fashion. Together, the data are consistent with the hypothesis that somatomedins are involved in developmental regulation of erythropoiesis.     

Effect of bone morphogenetic protein-6 on haemopoietic stem cells and cytokine production in normal human bone marrow stroma

Normal human bone marrow stroma cells include stem cells for both haemopoietic and osteochondrogenic lineages and express both bone morphogenetic protein (BMP) type I and type II receptors. As a member of the TGF-beta super-family, BMP-6 binds to both BMP type I and type II receptors and is involved in the developmental processes of renal and hepatic systems as well as of human foetal intestine. Also, BMP-6 induces osteoblastic differentiation of pluripotent mesenchymal cells and is an autocrine stimulator of chondrocyte differentiation. The present study was carried out to investigate the effect of BMP-6 on human cobblestone-area-forming cells (CAFC), that represent the functional primitive repopulating haemopoietic stem cell in long-term bone marrow culture. Also, the effect of BMP-6 on marrow stroma production of interleukin-6, -11 and their common receptor gp130 that is expressed in haemopoietic stem cells and is indispensable for their proliferation and tri-lineage differentiation was examined. Moreover, the effect of BMP-6 on marrow stroma release of soluble adhesion molecule VCAM-1 mediating the primitive haemopoietic stem cell adhesion to marrow stroma was examined. The number of CAFC was significantly reduced after BMP-6 treatment from 88+/-10 per 10(5)cells in control cultures in a dose dependent manner to only 48+/-3 per 10(5)cells in 50 ng/ml BMP-6-treated cultures, P< 0.01. Quantitative ELISA measurement revealed 50 ng/ml BMP-6 was able to significantly reduce IL-6 and IL-11 production from marrow stroma, P< 0.01. Also, BMP-6 significantly increased soluble gp130 release by 7.4-fold in 50 ng/ml BMP-6-treated marrow stroma cultures. The profound rapid increase in this natural antagonist of human IL-6 cytokine family may reduce the gp130 signaling. Also, the soluble VCAM-1 released increased by two-fold in 50 ng/ml BMP-6-treated marrow stroma cultures. The marked increase in the soluble form may exert an antagonist effect on the function of VCAM-1 (ligand for VLA4). Recently, blocking the VLA4/VCAM-1 adhesion pathway was shown to mobilise haemopoietic CD34 positive cells in normal individuals. Also, we previously observed a significantly lower expression of VLA4 (CD49d) on G-CSF-mobilised blood CD34 positive cells than on bone marrow CD34 positive cells before mobilisation in the same normal donors. Since BMP are currently being used in clinical trials for bone repair and fracture healing, the present results suggest a possible role for BMP-6 in mobilising CD34 positive cells for transplantation. Further in vitro tests are required to evaluate this potential mobilising role of BMP-6 in human long-term bone marrow culture.     

Extracellular bone matrix-derived growth factor

Demineralized extracellular bone matrix, when implanted subcutaneously into allogeneic rats, induces an invariant sequence of events resulting in de novo cartilage, bone and bone marrow formation. We have recently demonstrated the dissociative extraction and successful biological reconstitution of boneinducing molecule(s) in demineralized bone matrix. As mesenchymal cell proliferation precedes differentiation of endochondral bone, we have examined the bone-inductive molecules for mitogenic activity on human and rat fibroblasts and bovine endothelial cells. The results revealed that the molecular fraction obtained by molecular sieve chromatography on Sepharose CL-6B responsible for endochondral bone induction is capable of stimulating human and rat fibroblasts proliferation by 2500 and 300 % respectively, as compared to cells grown in a serum-free medium. Endothelial cells do not seem to share this response. This factor exhibits a significant effect on growth-promoting activity for both human and rat fibroblasts at a dose of 0.5 ng protein/ml of culture medium. These results demonstrate the presence of a tightly bound growth factor in the extracellular bone matrix.     

In vitro neurite extension by granule neurons is dependent upon astroglial-derived fibroblast growth factor

When grown in the absence of astroglial cells, purified mouse cerebellar granule neurons survive less than 36 hr and do not extend neurites. Here we report that low concentrations of basic fibroblast growth factor (bFGF, 1-25 ng/ml) maintained the viability and promoted the differentiation of purified granule neurons. The effect of bFGF on granule cell neurite outgrowth was dose dependent. Neurite outgrowth was stimulated markedly in the presence of 1-25 ng/ml bFGF, but effects were not seen below 1 ng/ml or above 50 ng/ml. When affinity-purified antibodies against bFGF (1-5 micrograms/ml) were added either to purified granule cells or to co-cultures of neurons and astroglial cells, process extension by granule neurons was severely impaired. The inhibition of neurite outgrowth in the presence of anti-bFGF antibodies was reversed by the addition of 25 ng/ml of exogenous bFGF. In addition to neuronotrophic effects, bFGF influenced the rate of growth of the astroglial cells. This result depended on whether the astroglia were grown in isolation from neurons, where low doses of bFGF (10-25 ng) stimulated glial growth, or in coculture with neurons, where much higher doses of bFGF (100-250 ng/ml) were needed for glial mitogenesis. Immunoprecipitation of lysates from 35S-labeled cerebellar astroglial cells with anti-bFGF antibodies revealed a single band after SDS-PAGE at 18,000 Da, the molecular weight of bFGF. These results indicate that glial cells synthesize bFGF and are possibly an endogenous source of bFGF in cerebellar cultures. Thus, astroglial cells synthesize soluble factors needed for neuronal differentiation.     

Stimulation of macrophage growth and multinucleated cell formation in rat bone marrow cultures by insulin-like growth factor I

In this study the effects of rhIGF-I on macrophage differentiation and growth have been studied using liquid suspension cultures of rat bone marrow cells. IGF-I stimulated macrophage growth in a dose-dependent manner, a maximum response was found at a concentration of 20 ng/ml. IGF-I effects could be ascribed to stimulation of both postmitotic and proliferating cells. A remarkable finding was that IGF-I induced formation of multinucleated cells (MNC). The MNC resembled macrophage-like cells (AcP, NSE positive). A monoclonal antibody to rhIGF-I significantly inhibited IGF-stimulated macrophage growth and MNC formation. A specific antibody to mouse CSF-1 reduced IGF-stimulated macrophage growth in mouse bone marrow cultures indicating that IGF-I effects could, at least in part, be ascribed to endogenous production of CSF-1. These findings indicate that IGF-I in concert with locally induced CSF-1 can influence the differentiation and growth of bone marrow-derived macrophages.