Vigorous neuronal differentiation of amplified and grafted basic fibroblast growth factor-responsive neurospheres derived from neuroepithelial stem cells

Neuroepithelial stem cells (NESCs) have emerged as a possible donor material aimed at neural transplantation for the repair of damaged neural circuitry, particularly because of their propensity to differentiate into neurons. We previously ascertained in vitro that NESCs derived from rat early embryos could be amplified in culture containing basic fibroblast growth factors (bFGF), and that neurospheres grown for 7 days in the culture had a strong tendency to differentiate into neurons. In this report, we analyze immunohistochemically the biological nature of bFGF-responsive neurospheres derived from NESCs. We first succeeded in amplifying the number of NESCs from the mesencephalic neural plate of embryonic day 10 Wistar rats with the addition of bFGF. Grown neurospheres were labeled with bromodeoxyuridine (BrdU) in vitro and were stereotactically transplanted into the right striatum of the normal adult Wistar rat. Two weeks after transplantation, a viable graft in the host brain was observed. While many BrdU/Hu double positive cells were seen in the graft, and a few BrdU/nestin double positive cells were also seen, no BrdU/GFAP double positive cells could be identified. These results suggested that bFGF-responsive neurospheres derived from NESCs demonstrated a propensity to differentiate into neurons in the adult brain environment. Furthermore, following in vitro amplification of the original stem cell number with bFGF, the grown neurospheres preserved their propensity to differentiate vigorously into neurons. NESCs are thus suggested as a feasible candidate for intracerebral grafting donor materials aimed at reconstruction of damaged neural circuits.     

血管内皮生长因子和碱性成纤维细胞生长因子对大鼠外周血内皮祖细胞培养的影响

目的 探讨不同培养条件对大鼠外周血来源内皮祖细胞（EPC）生长情况的影响。 方法 密度梯度离心法获得大鼠外周血单个核细胞,根据培养基中是否添加血管内皮生长因子（VEGF）、碱性成纤维细胞生长因子（bFGF）及培养板是否预铺纤连蛋白（FN）分组培养。观察记录细胞生长情况并进行统计分析,以免疫组化和免疫荧光法进行鉴定。 结果 大鼠外周血来源的单个核细胞在体外呈现贴壁生长,培养第7天各组细胞数及细胞集落数提示,在相同培养条件下,预铺FN可以促进EPC的贴壁增殖（t = 4.43,P < 0.05;t = 3.70,P < 0.05）。在同样预铺或未铺FN的情况下,在培养液中加入生长因子可促使单个核细胞更好地向EPC分化（t = －10.96,P < 0.01;t = －13.22,P < 0.01）。免疫组化及免疫荧光结果显示,细胞培养第4、7、10天,细胞表面CD34、CD133表达不断增强[（35.7±4.2）%、（60.1±3.8）%、（81.8±6.4）%;（3.2±0.9）%、（18.4±7.3）%、（32±3.8）%],第14天下降[（32.1±5.4）%,（1.9±2.7）%];而Flk-1表达在第4、7、10、14天均不断增强[（31.2±3.5）%、（40.6±5.3）%、（71.2±8.4）%、（81.5±4.1）%]。 结论 FN有利于内皮祖细胞的贴壁生长和增殖。VEGF及bFGF促进其增殖分化。内皮祖细胞的体外成功培养将为其应用于血管组织工程提供足够数量的种子细胞来源,并为外周血干细胞移植治疗多种疾病提供新的思路。     

Skull bone regeneration in primates in response to basic fibroblast growth factor.

OBJECT: The feasibility of using a biodegradable hydrogel incorporating basic fibroblast growth factor (bFGF) to induce bone regeneration at the site of a skull defect in monkeys was investigated. METHODS: Basic fibroblast growth factor was incorporated into a bioabsorbable hydrogel, which was prepared through glutaraldehyde crosslinking of gelatin. Following treatment of monkey skull defects measuring 6 mm in diameter (six defects/experimental group) with gelatin hydrogel incorporating bFGF, skull bone regeneration was evaluated using soft x-ray studies, dual x-ray absorptometry, and histological examinations. The water content of the hydrogels varied according to the glutaraldehyde concentration in the hydrogel preparation. Gelatin hydrogels incorporating 100 microg of bFGF significantly promoted bone regeneration and the skull defect was completely closed 21 weeks after implantation. This is in marked contrast with the effect of the same dose of bFGF in solution form. Bone mineral density (BMD) measured at the sites of skull defect was enhanced by the bFGF-incorporating hydrogels. The BMD enhancement was more prominent at lower water contents of hydrogel. Empty gelatin hydrogels neither induced nor interfered with skull bone regeneration. CONCLUSIONS: The findings of this study indicate that bFGF coupled with bioabsorbable hydrogel is a very promising tool to assist in the regrowth of bone at the site of a skull defect, which clinically has been recognized as almost impossible.     

Effect of prostatic growth factor,basic fibroblast growth factor,epidermal growth factor,and steroids on the proliferation of human fetal prostatic fibroblasts

To study the relationship between androgen metabolism and the pathogenesis of benign prostatic hypertrophy, we purified a growth factor from benign hyperplastic tissue of human prostates and assayed the proliferative responses of human fetal prostatic fibroblasts to the purified growth factor (hPGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), dihydrotestosterone (DHT), and estradiol (E₂). Prostatic tissue extracts were fractionated using heparin-Sepharose chromatography. The fraction that eluted with 1.3–1.7 M NaCl contained the majority of mitogenic activity. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) of the lyophilyzed active fraction showed a band at 17,000 daltons. Human prostatic fibroblasts were isolated from fetal prostate and tested for their proliferative responses to hPGF, bFGF, EGF, DHT, and E₂. hPGF, as well as bFGF and EGF, did increase tritiated thymidine incorporation into the cultured fibroblasts. DHT(10⁻⁷ M) had a significant stimulatory effect on cell growth in serum-free media after 6 days of culture. E₂(10⁻⁷ M) had no effect on cell proliferation. The combination of DHT and E₂ showed no synergistic effect. We conclude that our purified hPGF, bFGF, and EGF promote cell growth directly, DHT indirectly, while E₂ does not. The effect of DHT appears to be mediated via the increased production and/or secretion of growth factor(s). Possibly, the bFGF-like hPGF purified from human benign hyperplastic prostatic tissue is such a mediator. © 1996 Wiley-Liss, Inc.     

碱性成纤维细胞生长因子基因转染对鼠胎肝干细胞培养的优化作用

：目的 观察以重组腺相关病毒（rAAV）为载体介导碱性成纤维细胞生长因子（bFGF）基因转染对体外培养的鼠胎肝干细胞增殖及细胞周期的影响.方法 体外培养鼠胎肝干细胞,经rAAV作为载体介导bFGF基因转染,分为对照组、空载病毒转染组和bFGF转染组.用逆转录PCR和蛋白质印迹法检测鼠胎肝干细胞转染前、后bFGF基因和蛋白的表达.应用细胞生长曲线和CCK-8法观察细胞生长速度;采用流式细胞术测定细胞周期分布的变化.结果 转染bFGF后,bFGF转染组与对照组、空载病毒转染组相比,bFGF基因和蛋白水平均有表达,细胞的生长速度增快（q=20.43,P=0.001;q=26.52,P=0.001）,G0/G1期细胞减少,S期细胞增多（q=72.56,P=0.000;q=32.28,P=0.001）.结论 通过rAAV作为载体介导bFGF基因转染能促进体外培养的鼠胎肝干细胞增殖,对其培养有优化作用.     

Ontogeny of expression of basic fibroblast growth factor and its receptors in human fetal skin

The wounds in skin of early gestational fetus healwithout scar formation by a process resemblingregeneration rather than repair.1The ability torepair congenital anomalies in uterus such as cleft lipwith scarless healing will revolutionize the field ofreconstructive plastic surgery. Furthermore, if thebiological properties of scarless fetal healing aredetermined, these characteristics mightbe replicated inthe adult environment with tremendous clinicalbenefits. This non-scarring phenomenon is ge…     

Platelet-derived growth factor, basic fibroblast growth factor, and interferon gamma increase type IV collagen production in human fetal mesangial cells via a transforming growth factor-beta-dependent mechanism.

BACKGROUND: Glomerulosclerosis is characterized by glomerular accumulation of extracellular matrix following mesangial cell proliferation. The precise pathomechanism of glomerulosclerosis is still undetermined. Platelet-derived growth factor (PDGF) and basic fibroblast growth factor (b-FGF) are known to be mitogenic for mesangial cells, and interferon gamma (IFN-gamma) is known to have an inhibitory effect on mesangial cell proliferation. We attempted to clarify the role of these cytokines on mesangial matrix production using cultured human fetal mesangial cells (HMC). METHODS: HMC were incubated with these cytokines for 24-72 h and the levels of type IV collagen and TGF-beta in the cell supernatants were measured by enzyme immunoassay. RESULTS: PDGF, b-FGF, and IFN-gamma stimulated type IV collagen production by HMC in a dose- and time-dependent manner. The anti-TGF-beta neutralizing antibody clearly inhibited their stimulatory effect on type IV collagen production. PDGF and b-FGF also stimulated TGF-beta production by HMC in a dose-dependent manner, although IFN-gamma did not. CONCLUSION: PDGF, b-FGF, and IFN-gamma stimulate type IV collagen production in cultured HMC via a TGF-beta-dependent mechanism.     

Effect of maturation on the osteogenic response of cultured stromal bone marrow cells to basic fibroblast growth factor

Formation of bone-like tissue in culture by stromal bone marrow cells (SBMC) derived from young growing rats is dependent on dexamethasone (Dex) (Cell Tissue Res 254:317; 1988) and is significantly enhanced by basic fibroblast growth factor (bFGF) (J Bone Miner Res 8:919; 1993). The aim of this study was to examine the effect of maturation on the osteogenic potential and the response to Dex and bFGF of SBMC by using cultures derived from young growing (6 weeks old) and adult (9 months old) rats. SBMC cultures were grown in the presence of Dex (10⁻⁸ or 10⁻⁷ mol/L) at both P₀ and P₁ and either in the presence or absence of bFGF. The effect of Dex and bFGF on mineralized bone-like tissue (MBT) formation was assessed at P₁. The highest levels of mineralized tissue formation in P₁ subcultures in the absence of bFGF were obtained when cultures derived from young rats (6 weeks old) were treated with Dex 10⁻⁷ and 10⁻⁸ mol/L at P₀ and P₁, respectively, and when cultures derived from adult rats were exposed to Dex 10⁻⁸ mol/L both at P₀ and P₁. Under these optimal Dex concentrations, the amount of MBT formed by adult rat-derived cultures was 15-fold lower than that of young rat-derived ones. The addition of bFGF to P₀ cultures or to P₁ cultures grown under optimal Dex conditions enhanced MBT formation in P₁ cultures derived from both young and adult rats, but this effect was considerably more pronounced in the adult rat-derived cultures. The maximal levels of MBT formation were produced by cultures derived from adult rats treated with bFGF at both P₀ and P₁, whereas in cultures derived from young rats, the addition of bFGF at P₀ was not necessary for maximal MBT production. This stimulating effect of bFGF on MBT formation by adult rat-derived cultures was accompanied by a 2.2-, 1.8-, and 4.3-fold increase in proliferation, alkaline phosphatase activity, and Ca²⁺ deposition rate, respectively. bFGF increased the level of glucocorticoid receptor by approximately 2.3-fold in Dex-treated cultures derived from young animals. These results indicate that maturation is associated with a decrease in the proportion of osteoprogenitor cells in the stromal bone marrow and in their capacity to express the osteogenic phenotype. They further point to the significant role of bFGF in stimulating proliferation and osteogenic expression of stromal bone marrow osteoprogenitors derived from adult rats.     

A growth factor in bovine and human testes structurally related to basic fibroblast growth factor

Homogenates of human testes, epididymides and prostate, and calf testes and epididymides are mitogenic for cultured human foreskin fibroblasts. The growth factors appear similar in that they are inactivated by boiling and acid, but not by treatment with reducing agent. The growth factor in human and bovine testes was partially purified from tissue homogenates, prepared in high ionic strength buffer (pH 7.6) containing protease inhibitors, by ammonium sulfate precipitation and two cycles of heparin-Sepharose chromatography. The growth factor in calf testes was also partially purified from tissue extracted in ammonium sulfate without protease inhibitors, acidified to pH 4.5, and precipitated by ammonium sulfate followed by two cycles of heparin-affinity chromatography. A predominant 17,500 molecular weight (MW) growth factor was identified from alkaline homogenates of human and calf testes by its reactivity with antisera prepared against synthetic peptides whose sequences corresponded to residues 1-12 (amino-terminal), 33-43 (internal) and 136-145 (carboxy-terminal) of bovine basic fibroblast growth factor (bFGF). A slightly smaller 16,600 MW peptide from acidic extracts of calf testes also reacted with antisera to the three synthetic peptides. A 15,500 MW peptide, lacking immunoreactivity with antiserum to the amino-terminal synthetic peptide, was also seen. These findings suggest that a growth factor is present in human and calf testes that is structurally related to bFGF. The structure of the growth factors appears to be altered during the isolation procedure.     

碱性成纤维细胞生长因子在富集结直肠癌肿瘤干细胞中的作用

目的 研究不同浓度碱性成纤维细胞生长因子（bFGF）作用于结直肠癌细胞不同时间,富集肿 瘤干细胞的效果。方法? 结直肠癌DLD-1细胞分别在含5 ng/mL、10 ng/mL、20 ng/mL bFGF的无血清培 养基中悬浮培养,分为G1、G2、G3组,设置培养时间梯度为10 d、20 d、30 d,获得球细胞。采用流式 细胞术（FCM）检测细胞球中的CD44+、CD133+及CD44+CD133+双阳性的细胞表达比例,Real-time PCR 检测球细胞中的干细胞基因（KLF4、Nanog）;上皮间质转化基因（E-cadherin、Snail）;Wnt/β-catenine通路 基因（Wnt-3a）的 mRNA表达情况,成球实验检测细胞球的自我更新能力。结果 FCM检测结果：CD44+ 阳性表达的细胞表达以G2组20 d最高,CD133+及CD44+CD133+双阳性的细胞表达均以G2组20 d及G3 组10 d最高,差异有统计学意义（F=98.895、147.641、13.321,P<0.05）。 Real-time PCR检测结果：各组中 KLF4、Nanog、Snail以及Wnt-3a mRNA的表达均以G2组20 d表达最高（F=2.424、7.694、2.951、3.771, 均P<0.05）, E-cadherin基因G2组20 d mRNA表达最低（G2组30 d、G1组10 d除外）（F=10.620,P<0.05）。 成球实验结果：各组比较G1组20 d和G2组20 d成球数量明显多于其他组,差异具有统计学意义（F=14.279, P<0.01）;但 G1组20 d和G2组20 d比较,无统计学差异（t=0.605,P=0.578）。 结论 碱性成纤维细胞生长 因子无血清悬浮培养能够有效富集肿瘤干细胞,其中G2组培养20 d较其余组能更有效地富集结直肠癌肿 瘤干细胞。     

酸性成纤维细胞生长因子对人脐血内皮祖细胞的影响

目的 观察酸性成纤维细胞生长因子(aFGF)对体外培养内皮祖细胞(EPCs)数量、功能及凋亡的影响.方法 密度梯度离心法获取人脐血单个核细胞(MNCs),培养6 d后,收集贴壁细胞流式细胞仪和激光共聚焦显微镜鉴定EPCs.并向贴壁细胞分别加入aFGF 2、5、10μg/L干预培养24 h,同时对作用效果最为显著的组(aFGF 5μg/L组)分别培养6、12、24、48 h,分别观察EPCs数量、增生、迁移、黏膜及凋亡状况,从而对其时效关系进行观察.结果 与对照组比较,不同浓度的aFGF可以显著提高EPCs的数量、生物学功能并抑制其凋亡(P<0.05);本研究5μg/L aFGF作用24 h时对EPCs数量、生物学功能及凋亡抑制的影响最为显著(P<0.05).结论 aFGF增加体外培养条件下EPCs的数目、改善其生物学功能并抑制其凋亡.     

Expression of basic fibroblast growth factor in thyroid disorders

Morphologic and biologic studies were undertaken to clarify the biologic significance of basic fibroblast growth factor (bFGF) in human thyroid neoplasms. A total of 71 malignant tumors (50 papillary carcinomas, 14 follicular carcinomas, 7 anaplastic carcinomas), 11 follicular adenomas, 6 adenomatous goiters, and 6 Graves' disease tissues were examined employing immunohistochemical methods (avidin-biotin-peroxidase complex technique). An affinity-purified polyclonal rabbit antiserum to human bFGF was used as a primary antibody. The eluate of malignant thyroid tumor tissues from the heparin-Sepharose column was examined by Western blot analysis to elucidate the molecular weight form. With immunohistochemical staining, bFGF was frequently detected in the cytoplasm of malignant thyroid tumors compared to tissues of the benign diseases and normal controls. With anaplastic carcinoma, immunoreactivity of the tumor cells was particularly strong. In the correlative analyses between UICC TNM classification and bFGF staining in papillary carcinoma, there were significant differences when relating positive staining to the grade of nodal metastases. By Western blot analysis, the bFGF immunoreactivity was specifically detected in the two forms, with molecular weights of 18 and 33 kDa. The high-molecular-weight form was detected in only anaplastic carcinoma. The present investigations demonstrated a close correlation between the expression of bFGF and the degree of malignancy. bFGF might play an important role in promoting lymph node metastases. Moreover, the high-molecular-weight form of bFGF might have an intense influence on tumor growth.

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Resumen Se emprendió un estudio morfológico y biológico, con el propósito de clarificar la significación biológica del factor básico de crecimiento de fibroblastos (FBCF) en los neoplasmas de la glándula tiroides humana.Setenta y un tumores malignos (50 carcinomas papilares, 14 carcinomas foliculares y 7 carcinomas anaplásico), 11 adenomas foliculares, 6 bocios adenomatosos y 6 tejidos de glándula con enfermedad de Graves fueron examinados mediante métodos inmunohistoquímicos (técnica del complejo avidina-biotinaperoxidasa); se utilizó un antisuero policlonal purificado contra el FBCF humano, como anticuerpo primario. Además, se utilizó el análisis de Western blot para elucidar la forma de peso molecular.Con la coloración inmunohistoquímica, el FBCF fue detectado con frecuencia en el citoplasma de los tumores malignos de la tiroides, en comparación con lo observado en la enfermedad benigna y en los controles normales. La inmunorreactividad tumoral fue particulamente fuerte en el carcinoma anaplásico. En el análisis correlativo entre la clasificiación TNM UICC y la coloración del FBCF en el carcinoma papilar, se hallaron diferencias significativas relativas a la coloración positiva y al grado de la metástasis ganglionares.En el análisis Western blot, la inmunorreactividad FBCF fue específicamente detectada en las dos formas diferentes con pesos moleculares de 18 K y 33 K. La forma de alto peso molecular fue detectada sólamante en el carcinoma anaplásico.La presente investigación demuestra una estrecha correlación entre la expresión de FBCF y el grado de malignidad. El FBCF puede jugar un papel importante en promover las metástasis ganglionares. Además, la forma de alto peso molecular del FBCF puede tener una influencia más intensa sobre el crecimiento del tumor.

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Résumé Une étude morphologique et biologique a été effectuée pour clarifier la signification du facteur de croissance des fibroblastes de base (bFGF) dans les tumeurs de la thyroïde chez l'homme. On a étudié 71 tumeurs de la thyroïde (50 cancers papillaires, 14 cancers folliculaires, 6 adénomes solitaires, et 7 cancers anaplasiques, 11 adénomes folliculaires, 6 goitres adénomateux et 6 Maladies de Basedow) en utilisant des méthodes immunohistochimiques et notamment la technique du complexe avidinebiotine-peroxydase. On a employé un anticorps primaire fabriqué à partir d'un antisérum polyclonal de lapin purifié dirigé contre le bFGF humain. L'éluat des tissus thyroïdiens malins provenant de la colonne héparine-sepharose a été examiné selon la technique Western Blot pour déterminer son poids moléculaire. Le bFGF a été détecté plus fréquemment dans le cytoplasme des tumeurs thyroïdiennes malignes que dans les maladies bénignes et les contrôles. Dans le cancer anaplasique, l'immunoréactivité des cellules cancéreuses a été particulièrement forte. En ce qui concerne la corrélation entre la classification TNM de l'Union Internationale contre le Cancer et la coloration bFGF des cancers papillaires, il y avait des différences significatives correspondant au degré d'envahissement des ganglions lymphatiques. Dans l'analyse selon la technique du Western blot, l'immunoréactivité bFGF a été détectée spécifiquement sous les deux formes de bFGF ayant des poids moléculaires de 18 et de 33 K, respectivement. Le poids moléculaire de 33K n'a été détecté que dans le cancer anaplasique. Cette étude démontre la corrélation étroite entre l'expression bFGF et le degré de malignité. Le bFGF peut probablement jouer un rôle important dans la survenue de métastase lymphatique. Le type à poids moléculaire élevé de bFGF pourrait influencer d'advantage la croissance tumorale.

     

水通道蛋白和碱性成纤维生长因子在人腹膜间皮细胞的表达及意义

超滤衰竭是长期腹膜透析的主要并发症,其发生发展与腹膜结构、功能的变化密不可分.间皮细胞是腹膜滤过的主要屏障,且具有强大的分泌功能,在维持腹膜结构和功能的稳定方面起重要作用.     

碱性成纤维细胞生长因子对骨髓间充质干细胞扩增及分化潜能的影响

目的 观察碱性成纤维细胞生长因子(bFGF)对大鼠骨髓间充质干细胞(BMSCs)体外增殖及其分化潜能的影响.方法 体外培养大鼠BMSCs,分别设对照组和bFGF处理组,bFGF作用浓度分别为1、10、100 μg/L.作用72 h后,噻唑蓝(MTT)测吸光度(A)值反映细胞增殖.细胞免疫组织化学测细胞CD44的累积A值;逆转录-聚合酶链反应(RT-PCR)检测细胞CD44的mRNA相对表达量.结果 在1 μg/L bFGF作用下其MTT A值为0.334±0.036较对照组A值0.251 ±0.033明显增高(P＜0.05).在1 μg/L bFGF作用下其细胞免疫组织化学测得CD44的累积A值较对照组明显增高(P＜0.01);其RT-PCR测得CD44的mRNA相对表达量较对照组明显增高(P＜0.01).结论 作用浓度为1 μg/L的bFGF可促进BMSCs的体外扩增并有助于保持BMSCs的分化潜能.     

尿碱性成纤维细胞生长因子用于鉴别血管瘤和血管畸形的作用

目的 探讨尿碱性成纤维细胞生长因子（bFGF）用于鉴别血管瘤和血管畸形、判断血管瘤是处于增生期或消退期以及动态监测血管瘤病程的作用。方法 对133例患儿（包括增生期血管瘤69例、消退期血管瘤41例及血管畸形23例）,应用酶联免疫吸附试验,检测尿bFGF浓度,并以无上述血管病变的唇腭裂11例患儿作为对照。结果 增生期血管瘤患儿尿bFGF浓度显著高于消退期血管瘤、血管畸形患儿和对照患儿。前3者与对照患儿比较,差异均有统计学意义（P〈0．01）：后3者之间两两比较,差别均无统计学意义（P〉0．05）。结论 尿bFGF浓度有助于鉴别血管瘤和血管畸形;并可判断血管瘤是处于增生或消退期及动态监测血管瘤病程,可为分析血管瘤的发生机制提供依据。