酸性成纤维细胞生长因子及表皮生长因子对兔韧带细胞增殖的影响

目的：观察酸性成纤维细胞生长因子(aFGF)和表皮生长因子(EGF)对内侧副韧带(MCL)和前十字韧带(ACL)细胞增殖行为的影响。方法：培养10周龄新西兰白兔内侧副韧带和前十字韧带细胞，在培养液中分别加入aFGF和EGF，以XTT方法测定细胞的增殖行为。结果：aFGF在1ng／ml时即对两种细胞具有显著的促进增殖作用，其浓度达50ng／ml时，对MCL细胞的促进作用最大，达100ng／ml时对ACL细胞的促进作用最大。EGF在0．78ng／ml时即对MCL细胞有显著的促增殖作用，在1．56ng／ml时始对ACL细胞有显著的促增殖作用，其浓度达3．125ng／ml时对2种细胞的促进作用最大。aFGF和EGF在超过其最佳浓度后，随浓度升高促进作用均下降。结论：aFGF和EGF可以促进韧带成纤维细胞增殖。     

碱性成纤维细胞生长因子和表皮生长因子对骨骼肌源性干细胞的促增殖效应

目的观察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）单独及联合应用对骨骼肌源性干细胞（muscle derived stem cells，MDSCs）生长的影响。方法取出生24h内的昆明小鼠15只，采用连续预贴壁法从小鼠后肢肌分离培养MDSCs，用含2％胎牛血清的DMEM培养基促进其向骨骼肌细胞分化。取原代MDSCs及MDSCs分化细胞，采用免疫细胞化学染色检测干细胞标志Sca-1和骨骼肌细胞标志α-Sarcomeric肌动蛋白的表达。HE染色观察细胞肌管形成。MTT比色法检测不同浓度（6．25、12．50、25．00、50．00、100．00ng／ml）的bFGF和EGF单独应用96h对MDSCs增殖的影响以及二者（100．00ng／ml）联合作用24、48、72和96h对MDSCs增殖的影响。结果从新生小鼠后肢肌成功分离培养MDSCs，免疫细胞化学染色90％以上的MDSCs呈Sca-1阳性，分化形成的肌管呈α—Sarcomeric肌动蛋白阳性。HE染色可见肌管形成。bFGF、EGF对MDSCs的促增殖效应随浓度的增加而增加。与阴性对照组比较，bFGF于12．50ng／ml出现促增殖效应（P〈0．05）；25．00ng／ml组与12．50ng／ml组比较，作用提高（P〈0．01）；50．00、100．00ng／ml组较25．00ng／ml组无明显提升（P〉0．05）；EGF的作用与bFGF类似，但于50．00ng／ml时趋于饱和。与阴性对照组比较，EGF于72h、bFGF于96h表现促增殖效应（P〈0．01），而二者联合应用于24h即表现促增殖效应（P〈0．01），并于48、72和96h增殖效应均较单独应用显著提高（P〈0．05）。结论bFGF和EGF都能促进MDSCs的增殖，联合作用更快、更强。     

透明质酸联合碱性成纤维细胞生长因子对韧带细胞增殖的影响

目的 观察透明质酸（HA）和碱性成纤维细胞生长因子(bFGF）对内侧副韧带和前十字韧带细胞增殖行为的影响。方法 培养成年新西兰白兔内侧副韧带和前十字韧带细胞,在培养淮中分别加入HA、bFGF或两者的合物,以MTT方法测定细胞的增殖行为。结果 单独使用HA对前十字韧带细胞的增殖作用不明显,对内侧副韧带细胞的增殖有一定的促进作用。bFGF在1ng/ml时即对两种细胞显著的促增殖作用,其浓度达50ng/ml时,促进作用最大。100μg/mlHA与100ng/ml bFGF联合使用时,对两种细胞增殖的促进作用较单独使用HA或bFGF强。结论 HA可以作为bFGF的载体联合使用以促进韧带创伤愈合。     

成纤维细胞生长因子2在椎间盘中表达与作用的研究进展

成纤维细胞生长因子(fibroblast growth factor,FGF)目前发现总共有22种,但在椎间盘领域主要涉及的是成纤维细胞生长因子2(fibroblast growth factor 2,FGF2)[1]。因为FGF2呈碱性,故亦称为碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)。FGF2是一种具有促有丝分裂作用的活性多肽分子,在体内具有广泛的作用。在椎间盘中最基本的病理变化是椎间盘退变     

诱导成鼠骨髓源神经干细胞的实验研究

目的：探讨体外诱导成鼠骨髓源神经干细胞的可行性。方法：以含20ng／ml碱性成纤维生长因子(basic fibroblast growth factor，bFGF)与表皮生长因子(epidermal growth factor，EGF)的无血清培养液培养成鼠骨髓细胞，待细胞球形成后进行传代培养与单细胞克隆试验．以含100ng／ml bFGF培养液诱导分化，细胞免疫组化染色进行细胞鉴定。结果：骨髓细胞在诱导条件下形成表达Nestin抗原的细胞球．亦表达造血干细胞标志性抗原CD90。分离成单细胞后很快形成新的细胞球，并可分化出神经元与胶质细胞。结论：骨髓细胞可在体外诱导为能自我增殖的神经干细胞。     

碱性成纤维细胞生长因子促进冻干肌腱移植重建前交叉韧带后早期血管生成的组织学观察

目的观察碱性成纤维细胞生长因子（bFGF）促进冻干肌腱移植重建前交叉韧带（ACL）后早期血管生成作用。方法将100ng／mL的bFGF与冻干肌腱复合后，移植重建一侧犬ACL，另一侧单纯移植冻干肌腱做对照，14只家犬分别于术后1～6周取材，进行HE染色观察比较新血管的生成。结果实验组2～3周出现新生血管，4～5周达到高峰；而对照组4～5周出现新生血管。结论复合100ng／mL浓度的冻干肌腱移植重建ACL后，在新血管形成的时间及长入肌腱的深度方面优于对照组。     

三种生长因子对人胚半月板细胞增殖及细胞表型的影响

目的研究碱性成纤维细胞生长因子（basic fibroblast growth factor,bFGF）、转化生长因子β1（transforming growth factorβ1,TGF-β1）、胰岛素样生长因子1（insulin-like growth factor1,IGF-1）单独或联合作用于人胚半月板细胞,探讨半月板组织工程种子细胞大量扩增最佳作用组合及浓度。方法无菌条件下取健康妇女意外流产并自愿捐献的4个月人胚半月板,体外分离、培养半月板纤维软骨细胞,用Ⅱ型胶原免疫组织化学染色和Aggrecan免疫荧光染色检测其性状。取第3代细胞贴壁后使用血清饥饿法同步化细胞,加入IGF-1（1、10、50、100μg/L）、TGF-β1（0.1、1.0、5.0、10.0、50.0μg/L）、bFGF（5、10、50、100、200μg/L）作用于半月板细胞,每样本设8个复孔和阴性对照组,分别于作用后48h和72h采用MTT法检测软骨细胞增殖情况,以确定各生长因子最佳效应浓度。同法设7个组,每组8个复孔,分别为：阴性对照组、bFGF最佳效应浓度组（50μg/L）、TGF-β1最佳效应浓度组（5μg/L）、IGF-1最佳效应浓度组（50μg/L）、bFGF和TGF-β1最佳效应浓度联用组、bFGF和IGF-1最佳效应浓度联用组、TGF-β1和IGF-1最佳效应浓度联用组,48h和72h用MTT比色法得出吸光度（A）值并分析软骨细胞的增殖效应。结果第3代半月板细胞扩增前后细胞均表达Ⅱ型胶原和Aggrecan蛋白。48h和72h50μg/L IGF-1,5μg/L TGF-β1及50μg/L bFGF浓度组与对照组相比均具有促细胞增殖作用,且差异有统计学意义（P〈0.05）;此即为各生长因子最佳效应浓度。生长因子联用：bFGF50μg/L与IGF-150μg/L联用、IGF-150μg/L与TGF-β15μg/L联用具有协同效应,差异有统计学意义（P〈0.05）;bFGF50μg/L与TGF-β15μg/L联用无协同效应,差异无统计学意义（P〉0.05）。结论bFGF、TGF-β1、IGF-1单独使用均可体外扩增人胚半月板细胞,最佳效应浓度联用时bFGF/IGF-1、IGF-1/TGF-β1的扩增效果优于各自单独使用,可用于体外大量扩增种子细胞。     

生长因子对成人关节软骨细胞的促增殖作用

目的观察不同生长因子对成人关节软骨细胞（adult human articular chondrocytes，AHAC）增殖的影响，探索AHAC体外大量扩增的方法。方法以酶消化法从成人关节软骨分离细胞，条件培养基培养；传2代细胞分别用不同浓度成纤维细胞生长因子2（fibroblast growth factor-2，FGF-2）、转化生长因子β1（transforming growth factor β1，TGF—β1）、血小板衍生因子bb（platelet derived growth factor-bb，PDGF-hb）、肝细胞生长因子（hepatocyte growth factor，HGF）或其不同组合作用。用MTT法比较细胞增殖情况，用组化和免疫组化检测观察细胞表型变化。结果FGF-2、TGF—β1、PDGF—bb、HGF均有促AHAC增殖的作用，其最大效应剂量分别是50ng/ml、1ng／ml、1ng／ml、20ng／ml。5ng／ml FGF-2＋1ng/ml TGF-β1有最强的促增殖作用，继续加用PDGF—bb和（或）HGF无进一步促进作用；用这一因子组合培养AHAC，可以传10代以上，细胞扩增2000倍以上，且传9代细胞仍弱表达Ⅱ型胶原和aggrecan。结论FGF-2、TGF-β1、PDGF—bb、HGF均对AHAC有一定的促增殖作用；5ng／ml FGF-2＋1ng/ml TGF-β1有最大的促增殖效应，细胞短期内大量扩增，且在大量扩增的同时维持了一定的软骨细胞表型，因此是合适的AHAC体外大量扩增促进剂。     

探讨碱性成纤维细胞生长因子对肌瓣营养作用的机制

目的研究碱性成纤维细胞生长因子（basic fibroblast growth factor，bFGF）对失神经肌瓣营养的作用。方法将30只Wistar大白鼠，随机分为bFGF实验组，生理盐水对照组（对照组1）、bFGF全身用药对照组（对照组2）。建立腓肠肌瓣模型、保留动静脉带，离断支配神经然后行端端吻合，bFGF实验组术后从肌瓣基底留置硅管注射bFGF20 U（稀释于0．1ml生理盐水中），同时对照组1从硅管内注入0．1ml生理盐水，对照组2从健侧肌肉内注入等量bFGF20 U（稀释于0．1ml生理盐水中），每天1次，连续2周，分别于12周后行腓肠肌湿重、超微病理结构、肌瓣肌肉内神经组织化学染色检查。结果bFGF实验组各项指标明显优于生理盐水及bFGF全身用药对照组，差异有统计学意义，而生理盐水组与bFGF全身用药组比较，差异无统计学意义。结论持续增加局部bFGF浓度促进损伤神经的恢复，而加强肌肉营养防止肌萎缩。     

胸腔积液中成纤维细胞生长因子、转化生长因子-β和血小板衍生生长因子水平测定的临床意义

胸腔积液是临床上许多疾病的并发病，常见于感染、肿瘤、结缔组织疾病、心肝肾疾病等，由于在我国胸腔镜检查开展并不普遍，并且许多胸腔积液虽经胸腔镜检查也难以确诊，所以对胸腔积液的诊断与鉴别诊断目前仍主要依靠病史、体检、影像学检查、实验室检查来帮助诊断；血小板衍生生长因子(platelet-derived growth factor，PDGF)、成纤维细胞生长因子(fibroblast growth factor，FGF)、     

GDF‐5/7 and bFGF activate integrin α2‐mediated cellular migration in rabbit ligament fibroblasts

Cellular activities responding to growth factors are important in ligament healing. The anterior cruciate ligament (ACL) has poor healing potential compared to the medial collateral ligament (MCL). To assess the differences, we investigated the proliferation, migration, adhesion, and matrix synthesis responding to growth factors in rabbit ACL and MCL fibroblasts. ACL cell proliferation to basic fibroblast growth factor (bFGF), bone morphogenetic protein‐2, growth and differentiation factor (GDF)‐5, and GDF‐7 treatment was similar to that of MCL cells. GDF‐5 enhanced Col1a1 expression in ACL and MCL fibroblasts up to 4.7‐ and 17‐fold levels of control, respectively. MCL fibroblasts showed stronger migration activities in response to bFGF and GDF‐5 than ACL cells. GDF‐5/7 and bFGF also changed the stress fiber formation and cellular adhesion by modulating the distribution of integrin α2. Functional blocking analyses using anti‐integrin α2 antibodies revealed that cellular migration responding to growth factors depended on the integrin α2‐mediated adhesion on type I collagen. The expression of integrin α2 was also increased by growth factors in both cells. Our results demonstrate that GDF‐5/7 and bFGF stimulate cellular migration by modulating integrin α2 expression and integrin α2‐dependent adhesion, especially in MCL fibroblasts. These findings suggest that the different healing potential between ACL and MCL may be caused by different cellular behavior in the integrin α2‐mediated cellular migration in response to growth factors. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:225–231, 2010     

Differences in cellular properties and responses to growth factors between human ACL and MCL cells

The anterior cruciate ligament (ACL) has poor healing responses compared with those of the medial collateral ligament (MCL). It has been implied that this is partially due to the poor reparative capacity of ACL cells for ligament injury. The present study was designed to elucidate the reparative capacities of human ACL and MCL cells by investigating their cellular properties and their responses to growth factors. Human ACL and MCL were obtained from seven fresh human cadavers. The cells were isolated from each tissue, and primary cultures were used for the examination. The growth rates of all the human ACL cells were lower than those of the human MCL cells; consistent with this, the doubling time of the ACL cells was 30 ± 7.4% longer than that of the MCL cells. The chemotactic migration of human ACL cells was 33 ± 8.1% slower and the synthesis of DNA and collagen in human ACL cells was 29 ± 6.3% and 31 ± 9.7% lower, respectively, in comparison with those of MCL cells. Cellular responses, in terms of DNA synthesis, in human ACL cells to either basic-fibroblast growth factor (1.0 and 10.0 ng/ml) or transforming growth factor-β (1.0 ng/ml) were lower than those of human MCL cells. However, no differences in the cellular responses in terms of collagen synthesis were found. Composite data show that human ACL cells have poorer cellular properties and lower responses to growth factors compared with those of human MCL cells, which suggests that the reparative capacity of human ACL cells may be poorer than that of human MCL cells. Received for publication on Sept. 2, 1998; accepted on Jan. 29, 1999     

Intrinsic properties of anterior cruciate ligament (ACL) and medial collateral ligament (MCL) cells and their responses to growth factors (bFGF and TGF-β): An in vitro cell culture study

Different intrinsic properties of the constituent cells of the anterior cruciate ligament (ACL) and medial collateral ligament (MCL) have been proposed as factors involved in the differential repair mechanisms in these ligaments. To examine this hypothesis, we established primary cell lines from tissue explants. The outgrowth of cells from ACL explants was slower than that from MCL explants. The growth curves of ACL and MCL cultures at both passages 2 and 6 showed a slower rate of proliferation of ACL cells than of MCL cells (P<0.005). DNA synthesis supports the conclusion that these cells have differential proliferation rates in culture. An in vitro wound closure assay was performed by creating a uniform wound of 0.6-mm width in the confluent layer of ACL and MCL cultures. By 48h post-wounding, cell-free zones created in ACL cultures were, partially occupied by single cells in a non-confluent fashion. In contrast, the wounded zone in the MCL cultures was almost completely covered by the cells. Growth factors have been demonstrated to have both mitogenic and metabolic effects on cell cultures. We investigated the possible proliferative response of the ACL and MCL to growth factors, and we analyzed the in vitro effects of basic fibroblast growth factor (b-FGF) and transforming growth factor-β (TGF-β) on fibroblast cell cultures obtained from rabbit knee ligaments. Neither b-FGF nor TGF-β had any significant effect on the cell proliferation of ACL and MCL cultures after 48h. However, TGF-β did have an inhibitory effect on thymidine incorporation, especially at concentrations greater than 1 ng/ml, while b-FGF stimulated thymidine incorporation in ACL and MCL fibroblasts in a dose-dependent manner. b-FGF altered the cell phenotype to an elongated form, which suggests stimulation of cell migration. This in vivo, study implies a potential use for growth factors in the treatment of ACL and MCL injuries in vivo. Keynote Lecture, Japanese Orthopaedic Association, Chiba, Japan, June 30, 1995. Presented at the 21st Meeting of the Japanese Orthopaedic Society of Sports Medicine, Makuhari, Chiba     

Synovial fluid stimulates the proliferation of rabbit ligament. Fibroblasts in vitro.

This study was designed to test the hypothesis that synovial fluid may be inhibitory to cell proliferation. The effects of bovine synovial fluid (SF) and hyaluronic acid (HA) on the proliferation of normal rabbit medial collateral ligament (MCL), anterior cruciate ligament (ACL), and MCL scar cells were therefore investigated. Cell lines established from rabbit tissues were plated, incubated, and allowed to attach before treatment with varying concentrations of SF, HA, and a balanced salt solution (BSS). The BSS group was added as a control to observe the effects of media dilution alone on cell proliferation. Cell numbers from each group were quantified at 24, 48, 72, and 96 hours. Results showed that for all cell types, cell proliferation during the log phase of growth was significantly stimulated by SF. Maximum stimulation occurred in 20% SF with stimulation decreasing at higher concentrations of SF. HA had virtually no effect on scar and ACL cells, and only a slight stimulatory effect on MCL cells. Media dilution had no effect on scar cells and began to inhibit cell proliferation of ACL and MCL cells only at high dilutions. These findings suggest that low concentrations of bovine SF stimulate proliferation of rabbit ligament and scar fibroblasts in vitro by a mechanism that appears not to involve HA. Even in high concentrations, SF was not inhibitory to proliferation. The implications of these findings to ligament healing and normal ligament physiology require further investigation.     

Effect of basic fibroblast growth factor and hyaluronic acid on proliferation of rabbit chondrocytes in vitro

Objective: To investigate the effect of basic fibroblast growth factor (bFGF) and hyaluronic acid (HA) on the proliferation of rabbit chondrocytes in vitro. Methods: Chondrocytes from the knee joints of New Zealand white rabbits were cultured, bFGF or HA or both were added into the culture medium respectively, and the proliferation of the ehondrocytes was measured with MTT 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl-tetra-zolium bromide. (MTT, Sigma, M2128). Results: Basic fibroblast growth factor (10ng/ml) with low concentration of fetal bovine serum in the culture medium promoted the proliferation of chondrocytes significantly, and this effect reached its maximum when concentration of bFGF reached 50ng/ml. HA itself had no effect on the proliferation of chondrocytcs. However, when bFGF was used in combination with HA, especially when the concentration of bFGF was 50-500ng/ml and that of HA was 10-50ng/ml, the effect on the proliferation of chondrocytes was much more than when bFGF or HA was used alone. Conclusions: bFGF can promote the proliferation of chondrocytes. HA, which has no effect on the proliferation of the cells, can maintain a normal growth of chondrocytcs.When bFGF is used in combination with HA, more proliferation is obtained.     

碱性成纤维细胞生长因子对成骨细胞粘附特性影响的实验研究

目的 针对骨组织工程中成骨细胞与基质材料间促粘附问题,研究碱性成纤维细胞生长因子（ｂＦＧＦ）对成骨细胞粘附特性的影响。方法 取兔骨髓基质干细胞来源的成骨细胞体外培养,分别用５、１０、５０、１００及２００ｎｇ／ｍｌ浓度的ｂＦＧＦ诱导培养２４小时作为实验组;不含ｂＦＧＦ的培养基作为对照组。观察接种后０．５、１、２、４、及８小时各时间点成骨细胞粘附情况,体现学计量粘附细胞数量。结果 １０ｎｇ／ｍｌｂＦＧ