IGF-1信号通路相关蛋白在大鼠髁突软骨细胞增殖分化中的表达

目的:研究胰岛素样生长因子-1(IGF-1)信号转导通路在大鼠髁突软骨细胞增殖分化调控过程中相关基因的表达。方法:体外单层培养并鉴定出生后1、7、14、28 d共4组SD大鼠髁突软骨细胞。免疫组织化学方法检测细胞内IGF-1表达情况,Real-time PCR及Western印迹法检测细胞内IGF-1R、Bcl-2、Bax mRNA及蛋白表达。饥饿培养24 h后,实验组加入质量浓度为100 ng/mL的重组大鼠IGF-1细胞因子(rrIGF-1)继续培养24、48 h,CCK-8检测细胞增殖情况,Real-time PCR技术检测各组髁突软骨细胞内IGF-1R、IGF-2R、Raf1、GSK-3、IGFBP3、NF-κB、Bcl-2、Bax、integrin、TGF-βmRNA表达情况;对照组培养液不加rrIGF-1。结果:各鼠龄组SD大鼠髁突软骨细胞内IGF-1表达均阳性,IGF-1R mRNA及蛋白表达相对平稳,Bcl-2、Bax mRNA及蛋白表达逐渐增强,在出生14 d后表达下降(P<0.05)。加入100 ng/mL rrIGF-1后,髁突软骨细胞增殖速度显著增加,细胞凋亡减少,Real-time PCR结果显示实验组IGF-1R、GSK-3、Bax、integrin mRNA表达整体呈下降趋势;IGF-2R、Raf1、IGFBP3、NF-κB、Bcl-2、TGF-βmRNA表达水平总体呈上调趋势,差异有统计学意义。结论 :IGF-1介导的信号转导途径参与了髁突软骨细胞的增殖、分化以及软骨形成早期的调控,并可能与integrin、TGF-β等其他蛋白相互作用,共同参与髁突发育过程。     

Immunohistochemical localization of fibroblast growth factor-1 (FGF-l)and FGF-2 in cultured human ameloblastoma epithelial cells and ameloblastoma tissues

Fibroblast growth factor-1 (FGF-1) and FGF-2 are mitogenic polypeptides that may contribute to neoplastic cell proliferation. In the present study, we established a serum-free culture system for ameloblastoma cells and demonstrated that the addition of FGF-1 and FGF-2 enhanced cell growth in a dose-dependent manner. Immunoperoxidase staining of cultured cells demonstrated strong expression of FGF-1 and FGF-2. In tissue specimens, FGF-1 was localized in epithelial cell components of ameloblastomas, whereas FGF-2 was mainly found in the basement membranes with only moderate staining in epithelium. These data suggest that both FGF-1 and FGF-2 may contribute to the growth and development of ameloblastomas.     

The influence of masseter activity on rat mandibular growth

Many studies have shown that mandibular and condylar growth is affected by compressive forces on mandibular bone and the condyle. It has been reported that chondroblastic differentiation and proliferation in chondrocytes play important roles in condylar growth. However, the influence of reduced compressive force on chondroblastic proliferation and mandibular bone formation is not fully understood. Thirty-six 3-week-old male Wistar rats were used in this study. In the experimental group, the masseter muscles were bilaterally resected to evaluate the influence of masticatory force on mandibular and condylar bone morphology. Six weeks after the operation, while the rats were in the pubertal growth stage, lateral X-rays were taken to analyze the skeletal pattern of the mandible. The form of the condyle and the thickness of the chondroblastic layers were evaluated by toluidine blue staining. Chondroblastic proliferation was identified by insulin-like growth factor-1 receptor (IGF-1r) immunostaining and bone resorption of the condyle was assessed by measuring tartrate-resistant acid phosphatase (TRAP) activity. Lateral X-rays of the mandible showed that rats in the experimental group tended to have large mandibular plane angles. The chondroblastic layer in the condyles of the experimental group rats was thinner than in the control group. The expression of IGF-1r immunopositive cells in the experimental group was significantly lower than in the control chondrocytes, and the number of TRAP-positive cells was significantly higher in the condylar bone of the experimental group. We conclude that masseter muscle activity is closely related to mandibular morphology during growth.     

大鼠下颌功能前伸后激活髁突胰岛素样生长因子1基因表达

目的研究用功能矫治器前伸生长期大鼠下颌后，髁突软骨中胰岛素样生长因子（ＩＧＦ１）基因表达的变化规律，探讨功能矫形治疗的分子机理。方法将６０只雄性５周龄ＳＤ大鼠分为对照组和实验组，按１天、３天、１周、２周、３周和４周６个阶段，应用原位杂交技术研究髁突软骨中ＩＧＦ１ｍＲＮＡ的表达变化。结果正常生长期大鼠下颌髁突软骨有ＩＧＦ１ｍＲＮＡ表达；前伸大鼠下颌使髁突软骨细胞中ＩＧＦ１ｍＲＮＡ阳性强度和阳性细胞数目均增加。这一变化具有时间性，即实验３天后开始升高，实验后１周～２周达最高值，３周～４周时逐渐降低。结论功能前伸下颌后ＩＧＦ１ｍＲＮＡ转录升高，表明功能矫形后髁突软骨的生长改建可能是由于咀嚼肌牵张激活了ＩＧＦ１的基因表达。     

胰岛素样生长因子-Ⅰ及其与髁突软骨发育的调控

出生后的髁突软骨处于复杂的力学微环境中,其生长改建的生物学基础是髁突软骨细胞的增殖和分化。胰岛素样生长因子(IGF)-Ⅰ是髁突软骨生长发育过程中重要的生长因子之一。在细胞学水平,IGF-Ⅰ受胰岛素样生长因子连接蛋白(IGFBP)调控,通过与其受体的结合、激活来发挥生物学效应,其中包括调控细胞增殖、分化及程序性死亡,加快髁突软骨基质合成,参与髁突软骨细胞内力学信号的转导等。本文就IGF-Ⅰ及其受体,IGFBP及IGF-Ⅰ调控髁突软骨发育及其机制的研究作一综述。     

Immunocytochemical localization of fibroblast growth factor-1 (FGF-1) and FGF-2 in oral squamous cell carcinoma (SCC)

The localization of fibroblast growth factor-1 (FGF-1) and FGF-2 in human oral squamous cell carcinoma (SCC) was examined by immunohistochemical techniques using anli-FGF-1 and anti-FGF-2 monoclonal antibodies. Immunofluorescence staining of two oral SCC cell lines revealed that growing cancel-cells were intensely positive for both FGF-1 and FGF-2, but confluent cells showed a faint immunostaining. In addition, two molecular mass species of FGF-1(16 and 18 kDa) and one of FGF-2 (18 kDa) were identified by Western blot in cell extracts derived from growing SCC cells, but not from confluent SCC cells. The growing cell extracts significantly stimulated the proliferation of human umbilical vein endothelial cells. Immunoperoxidase staining of 13 oral SCC cases showed that both well-differentiated and poorly-differentiated cancer cells were positive for FGF-1 and FGF-2 with high frequency and intensity as compared to normal oral epithelium. These results indicate that SCC cells express high levels of endogenous FGF-1 and FGF-2, and suggest that these growth factors may contribute to cancer cell growth.     

Growth regulation of the rat mandibular condyle and femoral head by transforming growth factor-{beta}1, fibroblast growth factor-2 and insulin-like growth factor-I

The mandibular condyle is a major growth site and is known to adapt to functional factors. Numerous studies have been performed on the effects of growth factors on the metabolism of primary cartilages, but only a few investigations have examined their action on primary and secondary cartilages. Therefore, the purpose of this study was to compare the effects of insulin-like growth factor-I (IGF-I), transforming growth factor-beta(1) (TGF-beta(1)), and fibroblast growth factor-2 (FGF-2) on the growth of secondary cartilage from the mandibular condyle and primary cartilage from the femoral head of new-born rats. In addition, synergy between these growth factors was investigated. The level of glycosaminoglycan (GAG) and DNA synthesis was analysed after 5 days in culture with the growth factors. The effects of TGF-beta(1) and FGF-2 on growth, tissue organization, and the GAG and collagen content were also evaluated.The stimulation of cell proliferation by the growth factors was higher in the mandibular condyles than in the femoral heads. The content of the matrix components was reduced more by FGF-2 in the mandibular condyles than in the femoral heads. Both TGF-beta(1) and FGF-2 antagonized the stimulatory effects of IGF-I on GAG synthesis in the two types of cartilage. In contrast, the total growth of mandibular condyles was not affected by TGF-beta(1) and FGF-2 while that of femoral heads was strongly reduced. This was mainly due to the inhibition of chondrocyte hypertrophy. These results show that in spite of the extensive effects of growth factors on the metabolism of mandibular condyles, their dimensional growth was not affected.     

Regulatory effects of FGF-2 on the growth of mandibular condyles and femoral heads from newborn rats

The secondary cartilage of the mandibular condyle is considered to be adaptive to functional factors. In the last decades, growth factors have also been shown to be potent regulators of cartilage metabolism. Moreover, it has been suggested that growth factors may differentially regulate the growth of primary and secondary cartilages. However, only a few studies have made a direct comparison of the effects of growth factors on both cartilages. Therefore, the aim here was to compare the effects of FGF-2 on secondary cartilage of the mandibular condyle and primary cartilage of the femoral head from 4-day-old rats in vitro. Cartilages were cultured for 1, 7 and 14 days with 0 and 100 ng/mL FGF-2. We evaluated the effects of FGF-2 on growth, tissue organisation, DNA and glycosaminoglycan (GAG) synthesis and GAG and collagen content. With FGF-2, the morphology of the mandibular condyles changed and the GAG and collagen contents were reduced. However, the growth of the mandibular condyles was not affected. On the contrary, the growth of the femoral heads was strongly reduced due to an inhibition of chondrocyte hypertrophy. In both cartilages, FGF-2 stimulated DNA synthesis in short-term cultures and reduced it in long-term cultures. In conclusion, FGF-2 had a larger effect on the metabolism of the mandibular condyles as compared to the femoral heads. However, the growth of the femoral heads was strongly reduced while that of the mandibular condyles was not affected.     

Growth stimulation of mandibular condyles and femoral heads of newborn rats by IGF-I

Primary and secondary cartilage differ in embryonic origin and are generally considered to have a different mode of growth. However, few experimental studies exist that directly compare the two types of cartilage and their growth regulation. The regulation of cartilage growth is a complex mechanism involving growth factors like insulin-like growth factor-I (IGF-I). The purpose of this study was to compare the growth of mandibular condyles of 4-day-old rats with that of femoral heads in vitro and to analyze the effects of IGF-I. Explants were cultured for up to 2 weeks with 0, 5, and 25 ng/ml IGF-1. Both, 5 and 25 ng/ml IGF-I significantly stimulated growth of the mandibular condyles while only 25 ng/ml IGF-I stimulated growth of the femoral heads. IGF-I increased glycosaminoglycan synthesis of both condylar and femoral cartilage. However, only the DNA synthesis of the mandibular condyles was significantly increased by IGF-I while that of the femoral heads was not affected. It is concluded that IGF-I stimulates growth of both secondary condylar cartilage and primary femoral cartilage. The mandibular condyle appears to be more sensitive to IGF-I than the femoral head, which may partly be due to the different developmental stage.     

Immunohistochemical localization of fibroblast growth factor-1 (FGF-1), FGF-2 and fibroblast growth factor receptor-1 (FGfR-1) in pleomorphic adenoma of the salivary glands

Fibroblast growth faclor-1 (FGF-l) and FGF-2 are heparin-binding polypeplides that are potent mitogens for neoplastic cells. In this study, fibroblast growth factor-1 (FGF-l), FGF-2, and fibroblast growth factor receptor-1 (FGfR-1) were immunohistochemically analyzed in 10 patients with pleomorphic adenoma of the salivary gland by using specific monoclonal antibodies. The tumor tissues were histopathologically classified as: tubular, solid, myxoid or chondroid. Both FGF-1 and FGF-2 were immunohistochemically identified in the tumor cells of all histological types. In addition, immunoreactive FGF-2 was also found in the basement membrane of tubular type tumor cells. Conversely. FGfR-1-positive tumor cells were essentially confined to the tubular and solid areas of tumors. Tumor cells in the myxoid and chondroid areas were FGfR-1 immunonegative. These results suggest that the co-expression of FGF and its receptor appears to be related to the proliferative activity of tumor cells in the tubular and solid areas, whereas loss of FGF receptor expression may be associated with the differentiation of tumor cells into myxoid and chondroid tissue types.     

The effects of elevated fibroblast growth factor 23 on mandibular growth in rats

ObjectiveThe aim of this study is to elucidate the local effects of fibroblast growth factor 23 (FGF23) in on mandibular condylar growth in growing rats.DesignGrowing Sprague–Dawley rats received intra-temporomandibular joint injections of phosphate buffer solution (PBS), adenovirus-mediated green fluorescent protein (Ad-GFP) or adenovirus-mediated fibroblast growth factor 23 (Ad-FGF23), which were marked as groups A, B, and C, respectively. In vitro, we treated rat mandibular cartilage chondrocytes with PBS, Ad-GFP, and Ad-FGF23.ResultsThe mandibular condyles in group C grew smaller sizes than those in the other control groups due to significant differences among the experimental and control groups with the value of C–D, Q–R (P ≤ 0.05), accompanied by diminished bone mass of sub-cartilage condyles via micro CT analysis. Histologically, the length of the hypertrophic zone was diminished and was associated with decreasing chondrocyte proliferation in group C. Quantitative real-time PCR indicated significant decreases in the expression of chondrogenesis marker genes, including Type X collagen (Col X) and SRY-type box 9 (Sox 9). Moreover, elevated Ad-FGF23 suppressed chondrocyte proliferation and the expression of the chondrogenic differentiation markers Col X and Sox 9 of in vitro.ConclusionsLocal injection of FGF23 suppressed the development and decreased the bone mass of condyles through the decreasing the formation of condylar cartilage, specifically by regulating condylar cartilage cell viability and chondrogenesis expression.     

生长激素对牙和颅颌面结构发育的影响

生长激素是个体出生后生长发育的重要调节物质,能促进体内肌肉、骨骼和软骨等多种组织内细胞的增生和分化。即生长激素能调节与牙形成相关的上皮-间充质相互作用,调节成釉细胞、成牙本质细胞和成牙骨质细胞分化成熟,影响牙体硬组织结构的形成;参与调节破骨细胞骨吸收过程,影响牙萌出;通过胰岛素样生长因子-1调节髁突软骨细胞的生长分化,影响颞下颌关节的发育;直接或间接影响膜内成骨和软骨成骨,进而调节颌骨的发育,影响面部结构。     

FGF-2对体外培养人牙髓细胞FGFR和OPN表达活性的影响

目的:研究成纤维细胞生长因子-2(fibroblast growth factor-2,FGF-2)对体外培养的人牙髓细胞表面成纤维细胞生长因子受体(fibroblast growth factor receptor,FGFR)及骨桥蛋白(osteopontin,OPN)表达的影响。方法:以改良组织块培养法体外获得人牙髓细胞,采用Western blotting法检测不同浓度FGF-2作用下牙髓细胞FGFR的表达情况;应用Real Time-PCR法,检测不同浓度FGF-2作用下牙髓细胞OPN mRNA的表达。结果:与对照组相比,FGF-2在1～50 ng/mL浓度下均可促进牙髓细胞FGFR的表达(P<0.05),最佳显效浓度为10 ng/mL。FGF-2在1～50 ng/mL浓度下均可诱导牙髓细胞OPN mRNA表达(P<0.05),OPN mRNA的表达在10 ng/mL的FGF-2作用下达到高峰。结论:FGF-2可促进体外培养人牙髓细胞FGFR和OPN的表达,在牙髓牙本质复合体修复中可能发挥重要作用。     

Interleukin-1 beta affects cyclooxygenase-2 expression and cartilage metabolism in mandibular condyle

Extracellular matrix degradation in mandibular condylar cartilage is mediated by various cytokines in the temporomandibular joint (TMJ). Interleukin-1 beta (IL-1β) is detected in joint structures with pathologic status, and participates in catabolic action in the extracellular matrix. The purpose of this study was to investigate the effects of IL-1β on cyclooxygenase-2 (COX-2) expression and cartilage metabolism using cultured chondrocytes from mandibular condyle. Articular chondrocytes from the porcine mandibular condylar cartilage around the surface were cultured and treated with 0–10 ng/ml IL-1β or 0–1000 ng/ml prostaglandin (PGE₂) for 0–24 h. The mRNA levels of COX-2, MMP-1, -3, and -13 were evaluated by real-time PCR analysis. The protein levels of PGE₂ and MMPs were examined by ELISA and Western blot analysis, respectively. The expression levels of COX-2 and PGE₂ were enhanced by exogenous IL-1β in chondrocytes. The mRNA levels of MMP-1, -3, and -13 were up-regulated by PGE₂ treatment dose-dependently. It is shown that the expression of COX-2/PGE₂ was enhanced by IL-1β in articular chondrocytes from mandibular condyle, and that MMP-1, -3, and -13 were induced by PGE₂, suggesting that IL-1β-induced COX-2/PGE₂ play a crucial role in catabolic processes of mandibular condylar cartilage under inflammatory conditions.     

rhTGF-β1对大鼠髁突软骨细胞SZP表达的影响

目的:观察rhTGF-β1对大鼠髁突软骨细胞SZP表达的影响.方法:酶消化法获取大鼠髁突软骨细胞,甲苯胺蓝和Ⅱ型胶原染色鉴定.细胞培养液中分别加入10 μg/L、20μg/L rhTGF-β1后,不同时间点应用RT-PCR和ELISA法检测SZP mRNA和蛋白的表达.结果:加入10μg/L、20μg/L rhTGF-...     

Distribution of fibroblast growth factors (FGFR-1 and -3) and platelet-derived growth factor receptors (PDGFR) in the rat mandibular condyle during growth

Authors – Visnapuu V, Peltomäki T, Rönning O, Vahlberg T, Helenius H Objectives – To elucidate the role of the fibroblast growth factors 1 and 3 (FGFR‐1, ‐3) and the platelet derived growth factor (PDGFR) in the growth of the mandibular condylar cartilage in the rat. Setting and sample population – Institute of Dentistry and Department of Biostatistics, University of Turku, Turku, Finland. The material consisted of 1‐ to 21‐day‐old Long–Evans/Turku rats (total of 24 animals, three in each age group). Design – An immunohistological in vivo study combined with histomorphometry and biostatistical analysis. Experimental variable – The animals were killed with an overdose of carbon dioxide and thereafter decapitated. Heads were fixed in 4% paraformaldehyde, decalcified in 12.5% ethylenediaminetetraacetic acid, cut sagittally into two halves and sectioned sagittally at 6 μm. In order to detect FGFR‐1, ‐3 and PDGFR the sections were treated with H₂O₂/methanol (1:100), after which FGFR‐1 and PDGFR monoclonal and FGFR‐3 polyclonal antibodies were applied. The reaction products were visualized by using the Vectastain ABC Elite Kit using peroxidase substrate kit DAB as substrate. Negative and positive controls were also prepared. The sections were counterstained with hematoxylin. Outcome measure – In order to measure the depth of the cell layer labeled with FGF‐1, ‐3 and PDGF receptors, the condylar head was divided into four regions: anterior, superior, posterosuperior and posterior. The measurements were made perpendicular to the articular surface using a computerized image analysis system, the images being acquired by means of a microscope connected to a CCD camera. The mean of five equally distributed measurements of each region was used to indicate the depth of the cell layers secreting the receptors. Regression analysis was used to evaluate the association between the depth of the labeled cell layer in relation to total depth of the condylar head, as a function of age. Results – Our results show that the depth of the cell layer labeled for FGFR‐1, ‐3 and PDGFR increase significantly as a function of age in the mandibular condylar head of rats. Conclusion – Increase in the cell layer labeled for FGFR‐1, ‐3 and PDGFR occurs during the stage when the articular function of the mandibular condyle intensifies. FGFR‐1, ‐3 and PDGFR evidently have an important role in the growth regulation of the condylar cartilage during the most rapid growth period in the rat.     

慢性睡眠剥夺对大鼠髁突软骨的影响

目的： 建立大鼠慢性睡眠剥夺模型,观察大鼠颞下颌关节髁突软骨形态变化和软骨中IL-1β、TNF-α、IGF-1和VEGF的表达变化。方法： 将60只大鼠随机分为实验组、对照组和恢复组。利用改良多平台法（modified multiple platform method,MMPM）对实验组和恢复组大鼠进行1、2、3、4周的慢性睡眠剥夺。恢复组大鼠睡眠剥夺后正常笼养1周。利用H-E染色观察颞下颌关节髁突的组织结构变化,免疫组织化学法检测IL-1β、TNF-α、IGF-1和VEGF在髁突软骨内的表达水平。采用SPSS 23.0软件包对数据进行统计学分析。结果： 改良水平台法可以成功建立大鼠慢性睡眠剥夺模型。H-E染色观察到实验组髁突软骨出现纤维带分裂、细胞界限模糊,恢复组纤维裂隙减小或被纤维样组织占据。免疫组织化学结果显示,IL-1β、TNF-α在实验组中的阳性表达显著高于对照组（P<0.05）,恢复组表达显著低于实验组（P<0.05）。IGF-1和VEGF在实验组中的表达显著高于对照组（P<0.05）,恢复组1、2、3周IGF-1和VEGF的阳性表达显著下降（P<0.05）。结论： 慢性睡眠剥夺可引起髁突软骨内IL-1β、TNF-α、VEGF的表达增加,加重炎症表现。慢性睡眠剥夺可导致髁突软骨内IGF-1表达增加,发挥保护和促进软骨改建的作用。慢性睡眠剥夺停止1周后,各因子表达有所下降,髁突进行恢复性改建。     

Expression of insulin-like growth factor-1 and proliferating cell nuclear antigen in human pulp cells of teeth with complete and incomplete root development

Aim  To quantify the expression of insulin-like growth factor-1 (IGF-1) and proliferating cell nuclear antigen (PCNA) in human pulp cells of teeth with complete or incomplete root development, to support the specific role of IGF-1 in cell proliferation during tooth development and pulp reparative processes.
Methodology  Twenty six pulp samples were obtained from freshly extracted human third molars, equally divided in two groups according to root development stage (complete or incomplete root development). All samples were processed and immunostained to determine the expression of IGF-1 and PCNA in pulp cells. Sections were observed with a light microscope at 80× and morphometric analyses were performed to calculate the area of PCNA and IGF-1 immunostaining using digital image software. Mann–Whitney's test was used to determine statistically significant differences between groups ( P  < 0.05) for each peptide and the co-expression of both.
Results  Expression of IGF-1 and PCNA was observed in all human pulp samples with a statistically significant higher expression in cells of pulps having complete root development ( P  = 0.0009).
Conclusion  Insulin-like growth factor-1 and PCNA are expressed in human pulp cells, with a significant greater expression in pulp cells of teeth having complete root development.     

Gene expression profiling of mouse condylar cartilage during mastication by means of laser microdissection and cDNA array

Little is known about the mechanisms of mandibular condylar growth. In this study, gene expression in the mandibular condylar cartilage of young post-natal mice was monitored by means of a cDNA microarray, real-time PCR, and laser microdissection before and after the initiation of mastication (newborn, 7 days, 21 days, initiation of mastication, and 35 days). Insulin-like growth factor-1 (IGF-I), transforming-growth-factor-beta-2 (TGFbeta2), and aggrecan mRNAs were clearly expressed at 21 days, while the expression of osteopontin mRNAs was most clear at 35 days. Parathyroid-hormone-related protein (PTHrP), Indian-hedgehog (Ihh), and insulin-like growth factor-2 (IGF-2) mRNAs were clearly expressed during lactation (newborn and 7 days). Heat-shock-protein 84 (HSP-84) and heat-shock-protein 86 (HSP-86) were clearly expressed at 35 days. These results revealed that gene expression changed during mandibular condylar cartilage growth, and that, interestingly, these changes coincided with the initiation of mastication.