鸡胚胎原始生殖细胞的培养和传代

本文旨在探索鸡胚胎原始生殖细胞(Primordialgermcells,PGCs)培养、传代以及各种因素对PGCs培养的影响。实验结果表明,在添加10%的胎牛血清、2%的鸡血清、2mmol/LL谷氨酰胺、1mmol/L丙酮酸钠、5.5×10-5mol/Lβ巯基乙醇、10μl/ml非必需氨基酸、以及5ng/ml人干细胞生长因子(Humanstemcellfactor,hSCF)、10U/ml鼠白血病抑制因子(Mouseleukemiainhibitoryfactor,mLIF)、10ng/ml碱性成纤维生长因子(Fibroblastgrowthfactorbasic,bFGF)、0.04ng/ml人白细胞介素11(Humaninterleukin11,hIL11),10ng/ml胰岛素样生长因子(Humaninsulinlikegrowthfactor,hIGF)的高糖DMEM培养体系中,以多次离散法进行传代获得5-6代鸡胚胎PGCs,有效地维持了PGCs的未分化状态和正常二倍体核型,同时能够定向地诱导分化为神经细胞,具有作为多能性胚胎干细胞的特征     

bFGF和BMP-2联合应用对体外培养兔骨髓间充质干细胞增殖与骨向分化的影响

目的:研究碱性成纤维细胞生长因子(bFGF)和骨形成蛋白-2(BMP-2)联合应用对体外培养兔骨髓间充质干细胞(BMSCs)增殖与骨向分化的影响.方法:体外培养兔骨髓间充质干细胞,在第2代细胞培养液中加入不同浓度的bFGF和BMP-2,依据加入bFGF和BMP-2浓度的不同分为5个实验组(组1:80 ng/ml bFGF;组2:80 ng/ml BMP-2;组3:30 ng/ml bFGF 30 ng/ml BMP-2;组4:50ng/ml bFGF 50ng/ml BMP-2;组5:80ng/ml bFGF 80ng/ml BMP-2)和对照组(不加任何生长因子),采用绘制生长曲线,四唑盐比色法(MTT),碱性磷酸酶(ALP)活性检测法和碱性磷酸酶(ALP)免疫组化染色法比较各组间差异,观察不同浓度的bFGF和BMP-2联合应用对兔骨髓间充质干细胞增殖与骨向分化的影响.结果:与对照组相比,单独应用80 ng/ml bFGF可显著促进BMSCs的增殖,但对BMSCs的骨向分化显著抑制;单独应用80 ng/ml BMP-2对BMSCs的增殖和骨向分化均有促进作用;30ng/ml bFGF 30 ng/ml BMP-2、50 ng/ml bFGF 50 ng/ml BMP-2和80 ng/ml bFGF 80 ng/ml BMP-2可显著地促进BMSCs增殖和促进BMSCs的骨向分化,且呈正性剂量-效应关系,联合应用两种生长因子较二者单独应用促细胞增殖及骨向分化的效果更为显著.结论:一定浓度范围内,bFGF和BMP-2的联合应用促进BMSCs增殖的同时也促进其骨向分化,两者对BMSCs有明显的协同增强的生物学效应.     

人神经干细胞的体外生物学特性

本实验利用有丝分裂因子,体外诱导生成人神 经干细胞(NSCs),观察其生长特性并进行鉴定。取胎龄10-22周的大脑半球,分散细胞后种于添加表皮生长因子(EGF,20ng/ml)和/或碱性成纤维生长因子(bFGF,20ng/ml)的培养基中。利用免疫组织化学方法鉴定分化后的细胞类型。同时,进行细胞克隆分析、传代培养及端粒酶活性检测。结果显示:NSCs呈悬浮生长的干细胞球,其特异性抗原nestin阳性。NSCs具有增殖能力,可连续传代而不丢失其增殖和多分化潜能的干细胞特性。撤除EGF和bFGF的作用,细胞停止分裂,并分化为神经元、星形胶质细胞和少突胶质细胞。克隆分析显示NSCs生长呈密度依赖性。人NSCs表达较低的端粒酶水平,并随培养时间延长而下调。研究表明,利用有丝分裂因子,可在体外成功诱导生成人NSCs,其生长,分化受内外源因素的调节,相关的机制还有待阐明。     

粘着斑激酶在bFGF引起细胞迁移中的动态变化及意义

本文旨在观察不同浓度碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)引起体外培养的ECV-304细胞迁移时粘着斑激酶(focal adhesion kinase,FAK)的动态变化及FAK与细胞迁移的关系。建立体外培养的ECV-304细胞划痕损伤模型,观察经不同剂量(0、5、10、15 ng/ml)bFGF作用12-24 h内细胞迁移距离(电脑图像测定)和FAK蛋白含量(Western blot)、活性(免疫沉淀加Western blot)和mRNA(RT-PCR)的动态变化。用免疫细胞化学(ABC法)染色研究整合素α3表达。结果发现,低浓度(5 ng/ml)bFGF促进细胞迁移,FAK蛋白含量增加42.07±2.02％、活性增加71.37±1.85％,与对照组比,差异显著(P<0.05),并与迁移距离呈正相关(P<0.05)。高浓度(15 ng/ml)bFGF抑制细胞迁移,FAK的变化相反。FAK mRNA的变化比蛋白变化早出现6 h。与对照细比,各实验组整合素α3表达无明显差异。由此可见,不同剂量bFGF对ECV-304细胞迁移的双相调节作用与FAK含量、活性与mRNA表达呈正相关,FAK在bFGF引起的细胞迁移的信号转导途径中起着重要作用。     

肾小球系膜细胞株的建立及影响其生长的因素

利用肾小球细胞外生长能力的差异,从SD大鼠肾小球中成功分离肾小球系膜细胞(MC)后,探索了培养MC的适宜条件;观察了胰岛素、碱性成纤维细胞生长因子(bFGF)、内皮素(ET-1)和肿瘤坏死因子(TNFα)对MC增殖的影响。结果表明,四种因子均可显著促进MC增殖(P<0.01),其中bFGF作用最强。当胰岛素和bFGF、TNFα或低浓度ET-1(≤10~(-8)mol/L)共同作用于MC时,其促MC增殖显示正协同作用(P<0.01,P<0.05);与高浓度ET-1(≥10~(-7)mol/L)共同作用于MC时,呈负协同作用(P>0.05)。     

建立鸡EPGCs单细胞克隆株初探

目的探索鸡EPGCs单细胞克隆建立细胞系的可能性,并对其生物学特性进行鉴定。方法采取19期和28期的鸡EPGCs,体外培养传至第4代的细胞聚合体,用胰酶消化将细胞分散成单细胞悬液,将单个细胞接种至96孔板,每个孔内接种1个细胞,生长出的细胞集落用胶原酶消化传代,细胞化学法和免疫荧光法检测多向分化细胞的表面标志物;常规染色体核型检查。结果接种的288个单细胞中有9个扩增,其中7个传至第2代,2个传至第4代,克隆形成率为3.1%。扩增出的2~4代单细胞克隆能稳定增殖不分化,具有正常二倍性染色体核型、碱性磷酸酶阳性、阶段特异性胚胎抗原(SSEA)-1等特征性细胞表面标志物呈阳性;离体情况下具有形成类胚体和类上皮样细胞的能力。结论建立鸡EPGCs单细胞克隆细胞系是可行的,其生物学特性稳定。     

卵泡刺激素和表皮生长因子对小鼠精原细胞增殖的影响

利用生殖细胞-体细胞体外无血清共培养模型研究了卵泡刺激素(FSH)和表皮生长因子(EGF)对小鼠A型精原细胞增殖的影响。精原细胞在ITS培养液(添加胰岛素、转铁蛋白和亚硒酸钠的DMEM)中培养24h后进行c-kit免疫细胞化学鉴定和EGF及其受体(EGFR)免疫细胞化学检测,72h后测定其形成集落数的情况。结果表明:ITS培养液能维持生殖细胞的活性,增殖细胞核抗原(PCNA)的表达增高。A型精原细胞呈c-kit阳性,EGF和EGFR主要表达于精原细胞。单独的FSH(1～100ng/ml)或EGF(1～10ng/ml)显著促进精原细胞集落数的增加。此外,EGF(0.1ng/ml)联合FSH(10ng/ml)具有加性效应,但更高剂量的EGF(1～10ng/ml)则降低了FSH的刺激作用。结果说明FSH可联合适量的EGF促进精原细胞的增殖。     

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。     

骨髓基质干细胞的诱导分化及其治疗癫痫的实验研究

目的探讨骨髓基质干细胞诱导分化为神经元样细胞的方法及脑内移植治疗大鼠癫痫模型的作用。方法无菌条件下分离纯化BMSCs,用bFGF 10ng/ml、RA0.5μmol/L的DMEM/F12诱导72h后,部分用于免疫荧光检测nestin,其余的继续用bFGF 10ng/ml、RA 0.5μmol/L及神经营养因子NT-3 20ng/ml、BDNF 20ng/ml的DMEM/F12诱导4d,检测GAD67。皮下注射匹罗卡品建立癫痫大鼠模型,采用行为学分析筛选模型。通过立体定位仪,用微量注射器将BMSCs来源的神经干细胞和神经营养因子移植入癫痫鼠海马内,观察大鼠行为变化,存活2、4周后,心脏灌注取脑,冰冻切片免疫组化双标检测移植细胞的存活、迁移、分化情况。结果BMSCs诱导72h后,nestin表达阳性,4d后GAD67检测60%阳性。采用匹罗卡品造模,方法简便,但死亡率较高,仅15%-20%的动物造模成功。BMSCs源神经干细胞移植后可在海马内存活,向周围的脑区内迁移和整合。结论BMSCs源的神经干细胞在体外可诱导为γ-氨基丁酸能神经元并且对慢性癫痫有一定的治疗作用。     

鸡胚胎原始生殖细胞体外培养

以14-15期鸡胚血液为材料,采用Ficoll密度梯度离心方法,提取鸡胚胎原始生殖细胞(primordial germ cells,PGCs),在无基质细胞和基质细胞上分别进行体外培养。从实验结果可以看出：在含有胎牛血清(fetal bovine serum,FBS)、鸡血清(chicken serum,CS)、碱性成纤维细胞生长因子(bFGF)、人胰岛素样生长因子(hIGF-1)、小鼠白血病抑制因子(mLIF)和青,链霉素双抗的M199培养液中培养时,鸡PGCs最多能够存活4天：当采用细胞因子和5天鸡胚胎性腺基质细胞共培养时能存活23代且每代细胞增殖可达近10倍。提纯后的PGCs细胞冻存复苏后,经台盼蓝染色鉴定存活率可达80％左右。     

Basic fibroblast growth factor enhances the capacity of bone marrow cells to form bone-like nodules in vitro

The role of basic fibroblast growth factor (bFGF) in the proliferation and differentiation of rat bone marrow cells in culture was studied. bFGF stimulated [3H]thymidine incorporation into these cells by 4-fold at a concentration of 0.3 ng/ml and half-maximal effect was observed at a concentration of 15 pg/ml. In addition to its mitogenic effect, bFGF stimulated alkaline phosphatase activity by 3.6-fold. Continuous treatment with bFGF (for 21 days) resulted in a 6.3-fold increase in the culture dish surface area covered by bone-like mineralized tissue. Maximal bone-like tissue formation was observed in the presence of 3 ng/ml bFGF with half-maximal effect at a concentration of 0.3 ng/ml. These results indicate the possible role of bFGF in the proliferation of osteogenic rat bone marrow cells and their differentiation into cells of osteoblast-like phenotype.     

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.     

重组人干细胞因子在昆虫细胞中的高效表达

含信号肽的可溶性人干细胞因子（ｈＳＣＦ）ｃＤＮＡ 基因重组于杆状病毒转移载体ｐＶＬ９４１ 中,重组转移载体ｐＶＬ９４１ＳＣＦ与野生型苜蓿夜蛾核型多角体病毒（ＡｃＮＰＶ）ＤＮＡ 共转染草地夜蛾细胞Ｓｆ９ 后,通过体内同源重组构建了重组病毒ＡｃＮＰＶＳＣＦ。Ｓｏｕｔｈｅｒｎ 杂交表明重组病毒基因组中含有ｈＳＣＦ基因片段。重组病毒感染单层Ｓｆ９ 细胞后,表达产物分泌到胞外培养液中。用ＭＴＴ 比色法和ＴＦ１ 细胞株测定表达产物与ＩＬ３ 的协同效应,测得感染重组病毒的培养细胞第三天表达量为１９７０ ｕｎｉｔｓ／ｍ Ｌ培养液。Ｗｅｓｔｅｒｎｂｌｏｔｔｉｎｇ 分析可见分子量为１８ ×１０３ 、２０ ×１０３ 和２２ ×１０３ 三条带。     

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。     

体外培养条件下SCF、LIF与bFGF对昆明白小鼠精原干细胞增殖的影响

生长因子作为细胞体外培养和在体细胞生长及增殖必需的调节因子 ,一直被广泛的关注。业已证明干细胞因子(ＳｔｅｍＣｅｌｌＦａｃｔｏｒ,ＳＣＦ)、白血病抑制因子 (ｌｅｕｋａｅｍｉａｉｎｈｉｂｉｔｏｒｙｆａｃｔｏｒ,ＬＩＦ)和碱性成纤维细胞生长因子 (ＢｓａｉｃＦｉｂｒｏｂｌａｓｔＧｒｏｗｔｈＦａｃｔｏｒ,ｂＦＧＦ)具有刺激细胞增殖的作用[1～ 4] ,但大都是对单一因子进行研究。本实验探讨用这三种生长因子的不同组合观察对小鼠精原干细胞增殖的作用 ,以期定性和定量的探讨出体外培养初期三种因子对小鼠精原干细胞生长的影响 ,为小…     

Analysis of endothelial cell locomotion: Differential effects of motility and contact inhibition

Video microscopy and digital time-lapse recording were used to monitor locomotion and proliferation of bovine pulmonary artery endothelial (BPAE) cells cultured with varying concentrations of basic fibroblast growth factor (bFGF). Cell trajectories were reconstructed using a generalized nearest-neighbor algorithm and analyzed to determine how cell motility is affected by cell-cell collisions, cell divisions, and increasing cell density. The temporal evolution patterns of the average speed of locomotion for all cells in a culture were computed and the effects of varying bFGF concentrations were analyzed. Intermediate concentrations of bFGF (30 and 50 ng/mL) significantly increased the speed of locomotion above the levels we observed with 0 and 100 ng/mL concentrations of bFGF. Increases in cell density due to proliferation were immediately accompanied by a decrease in the average speed of locomotion of the cell population. Finally, the effect of bFGF concentration on the overall cell proliferation rates was assessed. With the addition of 30 or 50 ng/mL of bFGF to the culture media, the observed cell proliferation rates increased significantly. The proliferation rates decreased when the bFGF concentration increased to 100 ng/mL. These results show that bFGF concentrations that increase the motility of BPAE cells also increase the observed cell proliferation rates. (c) 1994 John Wiley & Sons, Inc.     

bFGF Promotes the Proliferation of Rat Peripheral Blood Mesenchymal Stem Cells Through β-Catenin Signaling

bFGF (basic fibroblast growth factor, bFGF) could promote the proliferation of bone marrow and cord blood mesenchymal stem cells. However, the effect of bFGF on the proliferation of peripheral blood mesenchymal stem cells (PBMSCs) needs further research. This study aimed to investigate the role of bFGF on the culture and expansion of PBMSCs in vitro. Firstly, arterial blood was collected from rats abdominal aorta. After mononuclear cells (MNCs) were separated with Ficoll separation fluid, MNCs were cultured in the DMEM medium without bFGF (served as control group) or with bFGF (10, 20 ng/mL, served as 10 or 20 ng/mL bFGF group). PBMSCs were obtained by adherent culture method. The third passage of PBMSCs was detected for the MSC surface markers and the effect of bFGF on the cell cycle of PBMSCs using flow cytometry. The effects of bFGF on colony formation, cell growth, and the expressions of cyclin D1, cyclin E, p21 and β-catenin were evaluated. PBMSCs showed no difference in morphology among the three groups. PBMSC clonies appeared 14 days after cultivation. Compared with the control group, the cell growth confluence of PBMSCs was obviously increased by 40% and 80% in groups treated with 10 ng/mL bFGF or 20 ng/mL bFGF respectively after culture of 21 days (all P<0.05). Compared with the group treated with 10 ng/mL bFGF, the confluence of PBMSCs in 20 ng/mL group was further increased by 28% (P<0.05). Cells of the third passage were positively stained for CD29 and CD90, while were negative for CD45. These results were consistent with the phenotypic characteristics of MSCs. Compared with the control group, the colony number of PBMSCs in the 10 ng/mL and 20 ng/mL bFGF groups was increased by 51% (P<0.05) and 92% (P<0.05), respectively. Compared with the 10 ng/mL group, the colony number of PBMSCs was further increased in 20 ng/mL group by 14% (P<0.05). The growth curve of PBMSCs showed that after 7 days of culture, the number of PBMSCs in 10 ng/mL bFGF group and 20 ng/mL bFGF group was increased by 41% (P<0.05) and 61% (P<0.05), respectively. Moreover, the cell number had a statistically significant difference between these two groups (P<0.05). Results from flow cytometry cell cycle showed that the numbers of PBMSCs in the G1 phase of experimental groups were significantly decreased as the concentration of bFGF increased when compared with the control group (P<0.05), whereas the number of PBMSCs in the S phase was significantly increased (P<0.05). Immunofluorescence experiments showed that, compared with the control group, bFGF significantly promoted the nuclear translocation and expression of β-catenin in PBMSCs. Compared with the 10 ng/mL group, the PBMSCs in 20 ng/mL bFGF group showed stronger nuclear translocation and expression of β-catenin. Western blot experiments showed that the levels of β-catenin and its target proteins cyclinD1 and cyclinE were significantly increased (all P<0.05), whereas expression of p21 was significantly decreased in PBMSCs in the bFGF groups in a concentration dependent pattern when compared with control group (P<0.05). The study firstly confirms that bFGF promotes the proliferation of PBMSCs by regulating the β-catenin signaling pathway, which may facilitate the aquisition of larger number of PBMSCs for stem cell engineering in vitro.     

Impact of peptide growth factors on the culture of small preantral follicles of domestic cats

Small preantral follicles (40 to 90 microm) of domestic cats were cultured in the presence or absence of epidermal growth factor (EGF), insulin-like growth factor I (IGF-I), and basic fibroblast growth factor (bFGF) for 5 d. The success of culture was estimated by in vitro incorporation of Brom-desoxyuridine (BrdU) into the oocytes and granulosa cells. Addition of EGF (4, 20, or 100 ng/ml) to the culture medium had no significant effect on the incidence of in vitro DNA synthesis. After supplementation with IGF-I and bFGF, BrdU-incorporation into the follicles and oocytes increased in correspondence to the concentration used, with 20 ng/ml IGF-I and 10 ng/ml bFGF giving the highest effect. In medium containing EGF, the IGF-I-induced increase in BrdU incorporation was suppressed, while the effect of bFGF was not decreased. Simultaneous addition of IGF-I and bFGF did not result in a further increase in DNA synthesis in the oocytes and granulosa cells. We conclude that bFGF mainly induces the proliferation of granulosa cells while IGF-I is involved in cellular activation of oocytes, which is modulated by EGF.     

Effect of hormones and growth factors on the proliferation of adult cricket neural progenitor cells in vitro

In the adult cricket brain, a cluster of neuroblasts produces new interneurons that integrate into the mushroom body (MB), the main associative structure for multisensory information of the insect brain. In previous study we showed the antagonist role of the two morphogenetic hormones, juvenile hormone (JH) and ecdysone, on the regulation of adult MB neurogenesis in vivo. In order to examine whether these hormones act directly on neural progenitor cells, we developed an organotypic culture of MB cortices. Cell proliferation was assessed by 5-bromo, 2'-deoxyuridine (BrdU) incorporation. We showed that JH increased mushroom body neuroblast (MBNb) proliferation, confirming the mitogenic effect of JH observed in vivo. By contrast, ecdysone did not affect the amount of BrdU-labeled nuclei, suggesting that the inhibitory effect observed in vivo probably proceeded from an indirect pathway. We then examined the role of growth factors known to stimulate neural stem cell/progenitor cell proliferation in vertebrates. As shown by calcium imaging, MBNb only expressed functional receptors for insulin whereas mature interneurons responded to IGF-I and bFGF. Both insulin (10 microg/ml) and IGF-I (10 ng/ml) enhanced MB progenitor cell proliferation in culture, although the insulin effect was more pronounced. This effect was abolished when an inhibitor of polyamine biosynthesis was present in the medium, suggesting a link between polyamines and the insulin signaling pathway. By contrast, bFGF (20-200 ng/ml) failed to stimulate MBNb proliferation. Our results point to conserved and divergent mechanisms between vertebrates and invertebrates in the regulation of adult neural progenitor cell proliferation.     

Opposite effects of bFGF and TGF-beta on collagen metabolism by human periodontal ligament fibroblasts

This study evaluated the effects of bFGF and TGF-beta, individually and combined, on cell proliferation and collagen metabolism. Primary human periodontal ligament cells were stimulated with two concentrations (1 and 10 ng/ml) of each growth factor, both individually and combined. Proliferation was determined by a commercial biochemical assay. Real time RT-PCR determined gene expression of MMP-1 and -2, collagen types I and III, TIMP-1, -2 and -3. Autocrine effects on synthesis of bFGF and TGF-beta were evaluated by ELISA. Only TGF-beta, either isolated or associated with bFGF, significantly increased cell proliferation. TGF-beta had anabolic effects, increasing expression of type I and III collagen as well as of TIMPs, whereas bFGF had opposite effects. When bFGF and TGF-beta were associated, the anabolic effects prevailed. Synthesis of TGF-beta was induced only by the association of lower concentrations of the growth factors, whereas there was a dose-dependent production of bFGF. It is concluded that bFGF had a predominantly catabolic effect, and TGF-beta exerted an anabolic effect on hPDL cells.