The mechanisms underlying fibroblast apoptosis regulated by growth factors during wound healing

While investigating the mechanisms underlying cell death during wound healing processes, we uncovered the pro‐apoptotic effects of basic fibroblast growth factor (bFGF) on granulation tissue fibroblasts following pretreatment with transforming growth factor (TGF)‐β1 in vitro. bFGF induced caspse‐3 activation and apoptosis in TGF‐β1‐pretreated granulation tissue‐derived fibroblasts (GF‐1) following bFGF treatment for 48 and 96 h. In contrast, fibroblasts that had been treated in the same manner and that originated from the uninjured dermis did not display apoptosis, indicating that the mechanisms underlying apoptosis events in fibroblasts that originate from normal dermal and wound tissues differ. In this process, we also found that bFGF inhibited Akt phosphorylation at serine 473 and induced a rapid loss of phosphorylation of focal adhesion kinase (FAK) at tyrosine 397 in pretreated GF‐1 cells, an event that coincided with the dissociation of phosphorylated FAK from the focal adhesions. Therefore, inhibition of survival signals relayed via the disrupted focal adhesion structures and inactivated Akt following bFGF treatment may lead to apoptosis in GF‐1 cells pretreated with TGF‐β1. Pretreatment of GF‐1 with TGF‐β1 followed by the addition of bFGF resulted in significantly greater inhibition of phosphorylation of Akt and FAK compared to treatment with TGF‐β1 or bFGF alone. The combinatorial treatment also led to proteolysis of FAK and inhibition of FAK and Akt protein expression in GF‐1 cells. These findings demonstrated a significant role for the two cytokines in apoptosis of granulation tissue fibroblasts during wound healing. In vivo studies also confirmed a marked decline in phosphorylation and protein expression of Akt and FAK in bFGF‐injected skin wounds. These results led to the hypothesis that temporal activation of TGF‐β1 and bFGF at the injury site promotes apoptosis in granulation tissue fibroblasts, an event that is critical for the termination of proliferative granulation tissue formation. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

碱性成纤维细胞生长因子及胰岛素样生长因子1对精原干细胞增殖凋亡影响的机制

文题释义：碱性成纤维细胞生长因子：是细胞生长和分化的重要调节因子,具有促血管生成、细胞增殖、细胞趋化、细胞迁移等活性,在细胞分化和机体发育过程中发挥重要作用。碱性成纤维细胞生长因子通过与细胞膜表面的特异性配体结合,进而引发细胞内的一系列级联反应,从而产生各种生物学效应。 胰岛素样生长因子1：是多功能细胞增殖调控因子,主要分布在肝脏中,其与胰岛素样生长因子2、胰岛素及其受体一起构成了胰岛素样生长因子家族,其对细胞生长和代谢具有多效作用。 背景：生长因子作为体外细胞培养和体内细胞生长及增殖必需的调节因子,一直被广泛的关注。 目的：探讨碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)与胰岛素样生长因子1(insulin-like growth facter,IGF-1)联合作用对小鼠精原干细胞增殖、凋亡的影响。 方法：从6-8 d龄昆明雄性小鼠睾丸内分离培养精原干细胞并进行鉴定。将精原干细胞接种于经丝裂霉素C处理过的胚胎成纤维细胞饲养层上,分组干预：对照组加入正常DMEM培养基进行培养;bFGF、IGF-1组分别加入含20 μg/L bFGF、20 μg/L IGF-1的DMEM培养基进行培养;bFGF+IGF-1组同时加入含20 μg/L bFGF及20 μg/L IGF-1的DMEM培养基进行培养。采用CCK-8、EDU染色法分别检测精原干细胞增殖活性,流式细胞仪检测精原干细胞生长周期和细胞凋亡情况,Western blot检测增殖和凋亡相关蛋白PCNA、Bax、Bcl-2的表达。 结果与结论：①与对照组比较,bFGF组、IGF-1组、bFGF+IGF-1组吸光度值显著升高,与bFGF组、IGF-1组比较,bFGF+IGF-1组吸光度值进一步升高(P < 0.05),EDU染色得到与CCK-8实验一致的结论;②bFGF+IGF-1组S+G₂/M期细胞比例明显高于其他3组(P < 0.05),IGF-1组、bFGF组S+G₂/M期细胞比例高于对照组(P < 0.05);③与对照组比较,bFGF组、IGF-1组、bFGF+IGF-1组凋亡细胞降低;与bFGF组、IGF-1组比较,bFGF+IGF-1组凋亡细胞进一步降低;④与对照组比较,bFGF组、IGF-1组、bFGF+IGF-1组细胞中Bax蛋白相对表达水平显著下降(P < 0.01),Bcl-2和PCNA蛋白相对表达水平均显著升高(P < 0.05)。与bFGF组、IGF-1组比较,bFGF+IGF-1组细胞中Bax蛋白相对表达水平进一步下降(P < 0.01),Bcl-2和PCNA蛋白相对表达水平进一步升高(P < 0.05);⑤结果表明,bFGF、IGF-1通过上调PCNA和Bcl-2蛋白的表达,下调Bax蛋白的表达,促进细胞增殖,抑制细胞凋亡,二者联合作用效果最佳。 ORCID: 0000-0001-5693-3713(李宏) 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

碱性成纤维细胞生长因子促进大鼠硬腭穿孔的愈合

目的:研究碱性成纤维细胞生长因子 (bFGF)对硬腭穿孔愈合的影响。方法:在大鼠硬腭部制作早期硬腭穿孔动物模型,外用bFGF治疗硬腭穿孔。采用肉眼观察、病理学检查的方法进行评价。结果:肉眼观察发现bFGF组伤口愈合率明显高于对照组(P＜0.01); 病理学检查表明bFGF能刺激肉芽组织的增长,成纤维细胞分裂增殖; bFGF组成纤维细胞的核仁形成区嗜银蛋白颗粒数目为3.73±0.52,对照组为2.11±0.31(P＜0.05)。结论: bFGF有助于肉芽组织的生长,具有显著促进硬腭穿孔创伤愈合的作用,有助于硬腭穿孔的修复。     

碱性成纤维细胞生长因子骨诱导作用的研究进展

介绍碱性成纤维细胞生长因子对骨干细胞、成骨细胞、破骨细胞和血管形成的可能作用机理,及其与其它生长因子之间的相互作用。对碱性成纤维细胞生长因子骨诱导作用的研究进展进行综述。     

Expression of vascular endothelial growth factor in exuberant tracheal granulation tissue in children

Prolonged tracheotomy and endotracheal intubation often induce symptoms of airway obstruction and delay decannulation and extubation. Bronchoscopic examination of patients undergoing these treatments usually shows the presence of exuberant (pseudopapillary or nodular) granulation tissue occupying the airway lumen. An immunohistochemical analysis was undertaken of vascular endothelial growth factor (VEGF) expression in exuberant tracheal granulation tissue (n=17) obtained from children treated with prolonged tracheotomy or endotracheal intubation. Increased levels of VEGF protein and mRNA were expressed mainly by tracheal epithelial cells that migrated to cover the granulation tissue and partly by pericapillary macrophages in this tissue, whereas normal tracheal epithelium did not express VEGF. The VEGF expression level correlated significantly with the severity of the exuberant granulation tissue response (p=0·0018). As VEGF induces angiogenesis and vascular permeability, characteristics of granulation tissue, and plays a pivotal role in granulation tissue development, enhanced VEGF expression may be involved in the development of exuberant tracheal granulation tissue. Copyright © 1999 John Wiley & Sons, Ltd     

纳米银抗菌水凝胶敷料联合重组人碱性成纤维细胞生长因子在踝部开放骨折创面的应用

背景:纳米银抗菌敷料和水凝胶是新型敷料,用于感染伤口的治疗.重组人碱性成纤维细胞生长因子是临床常应用于开放性伤口的活性多肽类药物.目的:探究外用重组人碱性成纤维细胞生长因子、纳米银抗菌水凝胶敷料在踝部开放骨折清创术后创面愈合中的应用.方法:收集踝部开放骨折清创术后患者99例,根据随机对照表分为对照组、试验A组和试验B组...     

碱性成纤维细胞生长因子促进血管性痴呆大鼠音猬蛋白的表达

目的:检测音猬蛋白(Shh)在血管性痴呆海马组织神经干细胞中的表达及碱性成纤维细胞生长因子(bFGF)对其的影响.探讨bFGF影响神经干细胞增殖分化的分子调控机制.方法:采用大脑中动脉栓塞(MCAO)制作大鼠血管性痴呆模型,免疫组织化学检测海马神经干细胞BrdU、Shh的表达及bFGF的作用.结果:脑缺血再灌注第3天缺血海马神经元BrdU阳性细胞较假手术组明显增多,第7天达高峰.Shh反应产物脑缺血再灌注第3天较假手术组增多,随缺血再灌注时间的延长逐渐增多,第14天开始减少.注射bFGF后BrdU、Shh的表达较模型组明显增加.结论:bFGF促进神经干细胞的增殖及缺血脑组织Shh的表达,提示bFGF促进神经干细胞增殖作用可能由Shh信号介导.     

碱性成纤维细胞生长因子与阿尔茨海默病

碱性成纤维细胞生长因子与阿尔茨海默病@朱大明$暨南大学生物工程研究所!广东广州510632 @洪岸$暨南大学生物工程研究所!广东广州510632 @林剑$暨南大学生物工程研究所!广东广州510632阿尔茨海默病;;成纤维细胞生长因子,碱性;;细胞;;细胞凋亡;;蛋白质类~~~~     

Basic fibroblast growth factor (bFGF) contributes to the enlargement of brown adipose tissue during cold acclimation

The contribution of basic fibroblast growth factor to brown adipose tissue (BAT) enlargement during cold acclimation was investigated using rat brown adipocytes in primary culture. After cold exposure (at 5° C) for 28 days, the level of bFGF messenger ribonucleic acid (mRNA) in BAT of cold-acclimated rats was markedly increased with the increase in the BAT weight. In addition, the blood plasma from cold-acclimated rats considerably enhanced the expression of basic fibroblast growth factor mRNA in rat brown adipocytes. Likewise, the blood plasma from cold-acclimated rats significantly stimulated the growth of rat brown adipocyte precursor cells compared with that from warm-acclimated rats, whereas there was no difference of effect between the two blood plasmas on the growth of bovine capillary endothelial cells. Basic fibroblast growth factor, but not platelet-derived growth factor stimulated the growth of brown adipocyte precursor cells. The conditioned medium from brown adipocyte primary culture markedly stimulated the growth of bovine capillary endothelial cells and the effect was inhibited considerably by antibasic fibroblast growth factor antibody. These results suggest that some factors concerned with the growth of brown adipocyte precursor cells are present in the blood plasma from cold-acclimated rats, and that basic fibroblast growth factor produced by brown adipocytes may significantly contribute to BAT enlargement by autocrine mechanisms during cold exposure.     

Basic fibroblast growth factor production in vitro by macrophages exposed to Dacron and polyglactin 910

Macrophage activation by implanted bood-contacting biomaterials modulates smooth muscle cell and endothelial cell ingrowth. The present study evaluates the in vitro interactions between Dacron or polyglactin 910 with macrophages derived from rabbits fed either normal or atherogenic diets. Peritoneal macrophages were cultured in the presence or absence (negative controls) of either biomaterial for 7 weeks. Conditioned media was evaluated for mitogenic activity using a rabbit aortic smooth muscle cell bioassay with or without preincubation with neutralizing anti-basic-FGF antibody. Results demonstrated increased mitogen release from macrophages harvested from the atherosclerotic rabbits. Only macrophages harvested from normal diet fed rabbits increased their mitogen release following exposure to either polyglactin 910 (p < 0.05) or to Dacron (p < 0.005) over controls. The stimulation of mitogen release by polyglactin 910 did not significantly exceed that in response to Dacron. In rabbits fed normal diets neutralization with the anti-basic-FGF antibody inhibited 100% of the Dacron induced mitogen release as compared to 36% of the polyglactin 910 induced mitogen release (p < 0.01). These results demonstrate significant induced mitogen release from macrophages exposed to biomaterials in vitro, much of the smooth muscle cell mitogen represented by basic-FGF.     

Purification and assay of intact human basic fibroblast growth factor using heparin-sepharose chromatography

Summary Intact human hepatoma derived basic fibroblast growth factor (bFGF) is a cationic 18 400-molecular-weight polypeptide, which stimulates the proliferation of 3T3 and endothelial cells at 1 ng/ml. bFGF has a strong affinity for heparin, and can be purified to homogeneity from human hepatoma cells using heparin-Sepharose chromatography. A three-step procedure is used: (a) extraction of the cells with 1M NaCl at pH 7.5; (b) Bio-Rex 70 cation exchange chromatography; and (c) heparin-Sepharose chromatography. The molecular weight of bFGF depends on the pH of the cell extraction step. When extracted at neutral pH, intact bFGF is obtained with a molecular weight of 18 400, however when extracted at pH 3.5 to 4.5 the molecular weight of bFGF is about 16 500. Lowering the molecular weight by acid pH extraction can be inhibited with a mixture of the proteinase inhibitors, PMSF, leupeptin, and pepstatin. These results suggest that degradation of bFGF is the result of cleavages by acid proteinases. It has been determined by amino acid sequence data and western blot analysis that the cleavages occur at the amino terminus of bFGF. The lower molecular weight form of bFGF has the same biological activity and exhibits the same chromatographic behavior on heparin-Sepharose as does intact bFGF.     

抗bFGF单克隆抗体的鉴定及其意义

用重组牛ｂＦＧＦ免疫Ｂａｌｂ／ｃ小鼠，通过细胞融合，以反向间接血凝和间接ＥＬＩＳＡ筛选，以及有限稀释法克隆化，建立了９株稳定分泌抗ｂＦＧＦ单克隆抗体（ｍＡｂ）杂交瘤细胞。对其中３株用Ｗｅｓｔｅｒｎｂｌｏｔ和生物活性抗体中和实验进行了鉴定。结果显示，ｍＡｂ可结合重组牛或人ｂＦＧＦ，并能抑制ｂＦＧＦ刺激Ｂａｌｂ／ｃ３Ｔ３细胞的生长；腹水的ＥＬＩＳＡ滴度为１∶３０００；用其中２株ｍＡｂ建立的双抗体夹心ＥＬＩＳＡ法灵敏度可达５０ｐｇ／孔。本文还讨论了抗ｂＦＧＦｍＡｂ的应用价值。     

碱性成纤维细胞生长因子诱导人间充质干细胞向心肌样细胞分化

目的研究碱性成纤维细胞生长因子(bFGF)在体外诱导人骨髓间充质干细胞(MSCs)向心肌样细胞分化中的作用,及其对MSCs增殖的影响。方法用含5-氮杂胞苷(5-aza)的培养基(A组)、含bFGF的培养基(B组)、含5-aza bFGF的培养基(C组)以及普通培养基(对照组)培养人骨髓MSCs。观察细胞形态的改变;免疫细胞化学法检测α-actin、cTnT和connexin-43的表达;RT-PCR法检测Nkx2·5、GATA-4和cTnT的mRNA水平;MTT法检测MSCs的增殖。结果A组和C组的部分细胞呈肌细胞样改变,表达蛋白α-actin,cTnT和connexin43;A组和C组的cTnT、Nkx2·5和GATA-4的mRNA水平明显高于对照组,B组的Nkx2·5和GATA-4的mRNA水平明显高于对照组;B组细胞增殖明显高于对照组,A组和C组细胞增殖明显低于对照组,但C组明显高于A组。结论bFGF能明显促进MSCs增殖,与5-aza合用能更好地诱导人MSCs向心肌样细胞分化。     

糖尿病小鼠视网膜碱性成纤维细胞生长因子的表达及细胞损害的电镜观察

目的：为临床研究提供形态学资料。方法：用免疫组化方法，观察碱性成纤维细胞生长因子(bFGF)在糖尿病小鼠视网膜的分布及表达密度；用透射电镜观察不同病程视网膜细胞的损害情况。结果：糖尿病及正常小鼠视网膜各层细胞均表达bFGF，反应强度和密度不同。发病组自6月龄起，居于大血管周围的阳性节细胞密度增加；不同病程bFGF的表达有不同程度的增加。随糖尿病病程延长，细胞超微结构受到不同程度的损害。结论：糖尿病时增多的bFGF直接或间接作用于血管内皮细胞和平滑肌细胞，刺激二者增殖，促进微血管病变的发生；另一方面，减弱视细胞与双极细胞间的信息传递，对糖尿病性盲的发生起重要作用。同时，各种细胞的超微结构及相应的功能也受到不同程度的损害。     

碱性成纤维细胞生长因子与卵巢癌的关系

目的　探讨碱性成纤维细胞生长因子 (basic fibroblast growth factor,b FGF)对卵巢癌细胞增殖、浸润和肿瘤血管生成的影响 ,及 b FGF单克隆抗体 (b FGF monoclonal antibody,b FGF- MAb)的治疗作用。　方法　将人卵巢癌细胞株 SKOV3接种于 2 4孔板 ,加入不同浓度的 b FGF,每日行结晶紫染色后测定光密度 (D4 90 )值 ,绘制细胞生长曲线 ;将 SKOV3细胞团接种于铺设有细胞外基质凝胶的 4孔板 ,每日测定癌细胞在凝胶中的浸润距离 ;建立 SKOV3细胞裸鼠皮下移植瘤模型 ,每周两次分别将 b FGF、b FGF-MAb和生理盐水注射于移植瘤周围 ,8周后测量肿瘤体积 ;对移植瘤组织切片行 因子的免疫组化染色、测定肿瘤内微血管密度 (microvessel density,MVD)。　结果　 b FGF能促进 SKOV3细胞增殖并呈浓度依赖 ,实验第 5天 ,5 ng/ml、10 ng/ml组细胞 D4 90 值是对照组的 1.0 9倍和 1.2 1倍 ;b FGF能促进 SKOV3细胞浸润并呈浓度依赖 (P<0 .0 5 ) ,第 7天 ,5 ng/ml、10 ng/ml组细胞浸润距离分别是对照组的 1.5 3倍和2 .4 5倍 ;b FGF组移植瘤体积和 MVD分别是对照组的 1.80倍和 1.4 6倍 (P<0 .0 5 ) ,b FGF- MAb组移植瘤体积和 MVD分别是对照组的 6 3.7%和 6 2 .8% (P<0 .0 5 )。　结论　b FGF能明显促进卵巢癌细胞的增殖、     

全反式维甲酸诱导骨骼畸形大鼠血清及羊水内碱性成纤维细胞生长因子的表达简

目的探究应用全反式维甲酸诱导骨骼畸形大鼠血清及羊水内碱性成纤维细胞生长因子（FGF2）的表达。方法选择孕10d的Wistar大鼠50只,体质量250～300g。分实验组和对照组.每组各25只。实验组予溶有全反式维甲酸的大豆油（40mg／mL）,按照135mg/hg以灌胃方式给药制作骨骼畸形胎鼠模型;对照组给予等体积的大豆油。孕20d时处死母鼠,采集母鼠血液,实验组每窝选择骨骼畸形胎鼠4只,对照组每窝随机选取胎鼠4只,抽取胎鼠羊水标本,应用酶联免疫吸附分析测定血液及羊水内FGF2浓度：利用游标卡尺测量胎鼠头臀长,双前肢各段及双后肢的长度。结果实验组胎鼠出现四肢发育不良、脊柱裂、下颌裂等畸形。实验组胎鼠头臀长、双前肢各段及双后肢长度与对照组相比.差异具有统计学意义（P〈0.05）。实验组母鼠血液及胎鼠羊水内FGF2的浓度与对照组相比[（24.124±1.271）pg／mL vs（27.451±2.026）pg／mL,（23.918±0.369）pg／mL vs（27.305±2.125）pg／mL],差异具有统计学意义（P〈0.05）。结论全反式维甲酸诱导骨骼畸形大隐.FGF2表茯情况低干骨骼发育正常的大鼠。     

Identification of basic fibroblast growth factor in papillary carcinoma of the thyroid

Basic fibroblast growth factor (bFGF) was identified in the papillary carcinoma of the human thyroid. Immunohistochemically, it was found that the reactivity for bFGF was localized in the cytoplasm of the neoplastic cells of the five papillary carcinomas. However the extract of the papillary carcinomas contained the mitogenic activity for endothelial cells. This bioactive molecule was determined as bFGF by using the heparin-Sepharose affinity chromatography and western blot analysis. The bFGF derived from human thyroid papillary carcinoma and the recombinant human bFGF stimulated the bromodeoxyuridine incorporation by the cultured human thyroid papillary carcinoma cells. These cells also showed positive staining for thyroglobulin and cytokeratin. These results indicate that bFGF, probably produced by the neoplastic cells, plays an important role in the development of papillary carcinoma of the thyroid with stimulation of angiogenesis as well as proliferation of the parenchymal cells.     

负压伤口治疗对糖尿病足创面肉芽组织血小板源生长因子表达的影响

目的探讨负压伤口治疗技术(NPWT)的应用对糖尿病足肉芽组织血小板源生长因子(PDGF)表达的影响。 方法选取解放军白求恩国际和平医院2010年1月至2014年12月收治的40例糖尿病足患者,采用单纯随机抽样法将患者随机分为NPWT组(20例)和对照组(20例),NPWT组创面采用负压伤口治疗技术治疗,对照组采用传统纱布换药治疗。两组分别于治疗前及治疗第12天取创面肉芽组织2份,一份置于4%多聚甲醛溶液中保存,行苏木精－伊红(HE)染色和免疫组织化学染色,HE染色观察肉芽组织形成情况,免疫组织化学染色观察肉芽组织PDGF的表达情况,通过Motic Medical 6.0数码医学图像分析系统检测吸光度值对PDGF蛋白行定量分析。同时,另一份肉芽组织标本－80℃冰箱中保存,采用Western blot法检测PDGF蛋白表达量。数据应用SPSS 13.0软件进行分析,PDGF吸光度值及PDGF蛋白表达量比较采用两独立样本t检验。 结果两组治疗前HE染色可见炎症细胞多,成纤维细胞、新生血管形成较少;两组治疗第12天肉芽组织HE染色均可见新鲜肉芽组织的形成,表现为新生血管、成纤维细胞增多,但与对照组相比,NPWT组新生肉芽组织增多较为明显。免疫组织化学分析显示,两组治疗后均可见PDGF蛋白表达上调;NPWT组治疗第12天PDGF吸光度值(0.41±0.05)与治疗前(0.29±0.04)比较,差异有统计学意义(t＝8.63,P<0.05);对照组治疗第12天PDGF吸光度值(0.33±0.04)与治疗前(0.28±0.04)比较,差异也有统计学意义(t＝3.62,P<0.05);但是,NPWT组治疗前后PDGF吸光度变化值为0.13±0.05,与对照组治疗前后PDGF吸光度变化值(0.05±0.03)比较,差异有统计学意义(t＝5.89,P<0.05)。Western blot分析显示,NPWT组治疗第12天PDGF蛋白表达量为0.24 ±0.04,与治疗前PDGF蛋白表达量(0.11±0.03)比较,差异有统计学意义(t＝11.01,P<0.05);对照组治疗第12天PDGF蛋白表达量为0.17±0.03,与治疗前PDGF蛋白表达量(0.12 ±0.03)比较,差异亦有统计学意义(t＝6.06,P<0.05);但是,NPWT组治疗前后PDGF蛋白表达量的变化值为0.12 ±0.03,与对照组治疗前后PDGF蛋白表达量的变化值(0.05 ±0.02)比较,差异有统计学意义(t＝9.06,P<0.05)。 结论应用NPWT可明显促进创面肉芽组织PDGF表达上调,从而促进创面的愈合。     

碱性成纤维细胞生长因子诱导非贴壁骨髓基质细胞向成骨细胞分化

目的：探讨碱性成纤维细胞生长因子(bFGF)对非贴壁骨髓前体细胞(NASP)的增殖分化作用。方法：分离骨髓前体细胞体外培养，用碱性磷酸酶化学染色及图象分析测定细胞克隆数；采用免疫组化(ABC法)进行骨钙素及碱性磷酸酶(ALP)测定，研究bFGF对NASP的促增殖作用。结果：bFGF能促进NASP细胞增殖，诱导NASP细胞产生碱性磷酸酶及骨钙素。结论：bFGF主要作用于非贴壁生长的基质前体细胞，bFGF能增加骨的数量，促进骨样组织形成。