生长因子网络调节对骨形成作用的研究Ⅴ.不同生长因子交互应用对人胚成骨样细胞增殖及分化的影响

目的：考察将转化生长因子β（ＴＧＦ－β）、胰岛素样生长因子Ⅱ（ＩＧＦ－Ⅱ）、碱性成纤维细胞生长因子（ｂＦＧＦ）及血小板衍生性生长因子（ＰＤＧＦ）４种生长因子两两交互应用对人胚成骨样细胞增殖与分化的影响。方法：在不同的实验间期检测成骨样细胞３Ｈ－胸腺嘧啶脱氧核苷、３Ｈ－脯氨酸的掺入量和碱性磷酸酶的含量。结果：ＰＤＧＦ、ｂＦＧＦ、ＩＧＦ－Ⅱ和ＴＧＦ－β４种生长因子交互联合应用,均对促进体外培养的人胚成骨样细胞的增殖和分化功能具有协同作用。结论：具有协同作用的生长因子联合应用,可提高其促进骨形成的生物效应。     

bFGF、IGF-Ⅰ及TGF-β1对人髁突软骨细胞增殖的影响

目的 研究胰岛素样生长因子（ｉｎｓｕｌｉｎ－ｌｉｋｅ ｇｒｏｗｔｈ ｆａｃｔｏｒ Ⅰ,ＩＧＦ－Ⅰ）,碱性成纤维细胞生长因子（ｂａｓｉｃ ｆｉｂｒｏｂｌａｓｔ ｇｒｏｗｔｈ ｂＦＧＦ）,转化生长因子β１（ｔｒａｎｓｆｏｒｍｉｎｇ ｇｒｏｗｔｈ ｆａｃｔｏｒ β１,ＴＧＦ－β１）单独及联合应用,对人髁突软骨细胞增殖的剂量－效应及时间－效应。方法 采用体外细胞培养技术及噻唑蓝比色法,对３种生长因子对软骨     

TGF—β1在C57BL／6N系小鼠腭突发育及腭裂形成中mRNA转录水平？…

目的 通过观察ＴＧＦ－β１在腭裂形成及腭突正常发育中的表达改变,探讨其在腭突不同生长时期的作用。方法 利用建立的腭裂模型,用原位杂交技术检测ＴＧＦ－β１在ｍＲＮＡ转录水平的变化。结果 ＴＧＦ－β１在正常组ＲＮＡ表达比相同腭突生长期的腭裂组明显增高（Ｐ〈０．０１）。正常组小鼠腭突发育至接触融合期,ＴＧＦ－β１ＲＮＡ表达与垂直期、上抬期之间也均有显著差异（Ｐ〈０．０１）。垂直期与上抬期之间无差异（Ｐ〉     

转化生长因子β对人牙周膜成纤维细胞的生物学作用

目的 研究转化生长因子β（ｔｒａｎｓｆｏｒｍｉｎｇ ｇｒｏｗｔｈ ｆａｃｔｏｒ－β,ＴＧＦ－β）对人牙周膜成纤维细胞（ｈｕｍａｎ ｐｅｒｉｏｄｏｎｔａｌ ｌｉｇａｍｅｎｔ ｆｉｂｒｏｂｌａｓｔｓ,ＨＰＤＬＦｓ）的生物学作用。方法 原代培养ＨＰＤＬＦｓ,并观察ＴＧＦ－β对ＨＰＤＬＦｓ的增殖、碱性磷酸酶（ａｌｋａｌｉｎｅ ｐｈｏｓｐｈａｔａｓｅ,ＡＬＰ）活性、骨钙素（ｏｓｔｅｏｃａｌｃｉｎ,ＯＣ）合成及矿化结节形成的作用。结果 ０．００５０～０．１０００ｍｇ／ＬＴＧＦ－β可明显刺激ＨＰＤＬＦｓ的增殖,０．００５０ｍｇ／ＬＴＧＦ－β可显著提高ＨＰＤＬＦｓ的ＡＬＰ活性,０．０００５～０．１０００ｍｇ／ＬＴＧＦ－β对ＨＰＤＬＦｓ合成ＯＣ和矿化结节的形成无明显影响。结论 ＴＧＦ－β对ＨＰＤＬＦｓ的作用呈剂量依赖性。ＴＧＦ－     

机械牵张作用对UMR—106细胞骨桥蛋白mRNA几TGF—β1 …

目的 探讨机械牵作作用对骨桥蛋白ｍＲＮＡ和转化生长因子β１ｍＲＮＡ表达的影响，说明正畸性骨改建的分子生物学机制。方法增益本外２细胞牵张施力模型，利用地高辛标记探针进行原位杂交，结合图象分析技术定量化研究基因表达的相对强度。结果 不同牵张强度和频率作用对ＯＰＮｍＲＮＡ和ＴＧＦ－β１ｍＲＮＡ表达影响的差别具有显著性。爸哟度－你物影响最为显著，２４ｈ时ＯＰＮｍＲＮＡ的表达强度从０．１６１３降低到０．１２     

白细胞介素－1β、转化生长因子－β对人牙周膜成纤维细胞DNA合成的影响

目的：观察白细胞介素－１β（ｉｎｔｅｒｌｅｕｋｉｎ－１β，ＩＬ－１β）和转化生长因子－β（ｔｒａｎｓｆｏｒｍｉｎｇｇｒｏｗｔｈｆａｃｔｏｒ－β，ＴＧＦ－β）单独作用、组合后对人牙周膜成纤维细胞（ｈｕｍａｎｐｅｒｉｏｄｏｎｔａｌｌｉｇａｍｅｎｔｆｉｂｒｏｂｌａｓｔ，ＨＰＬＦ）的ＤＮＡ合成量的影响。方法：用３Ｈ胸腺嘧啶脱氧核苷细胞内掺入法。结果：ＤＮＡ合成量显著增多。浓度为０．０１ｎｇ／ｍｌ的ＴＧＦ－β与浓度为１０ｕ／ｍｌ的ＩＬ－１β组合后对ＨＰＬＦ的ＤＮＡ合成量与单纯ＩＬ－１β组间无显著差异（Ｐ＞０．０５），而０．１ｎｇ／ｍｌ、１．０ｎｇ／ｍｌ的ＴＧＦ－β与三种浓度ＩＬ－１β组合后，ＨＰＬＦ的ＤＮＡ合成量显著高于单纯的ＩＬ－１β组。结论：ＩＬ－１β、ＴＧＦ－β对ＨＰＬＦ的ＤＮＡ合成有促进作用，这两种细胞因子组合并非各个细胞因子作用的简单相加     

大鼠下颌髁突软骨中胰岛素样生长因子Ⅰ及其基因的差异性表达——颅面生长伺服系统理论的直接证据

采用原位杂交和免疫组化技术，对１０只７周龄雄性ＳＤ大鼠双侧髁突中胰岛素样生长因子Ⅰ（ＩＧＦ－Ⅰ）及其ｍＲＮＡ表达进行研究。结果发现：生长期大鼠髁突软骨中ＩＧＦ－Ⅰ及其基因均有表达，但ＩＧＦ－Ⅰ阳性强度在生发层中最强，而ＩＧＦ－ⅠｍＲＮＡ在过渡层和成熟层中最强。表明髁突软骨具有局部合成和分泌ＩＧＦ－Ⅰ的能力，可介导生长激素的命令式作用，其差异性表达建立起局部反应的反馈环路，为生长伺服理论提供了直接证据     

功能矫形前伸大鼠下颌后髁突软骨β转化生长因子Ⅰ型受体(TβR-Ⅰ)表达变化的研究

目的：了解青春期ＳＤ大鼠髁突软骨β转化生长因子Ⅰ型受体（ＴβＲ－Ⅰ）在功能矫形前伸下颌后分布的变化，探讨ＴβＲ－Ⅰ与ＴＧＦ－β１表达的关系。方法：实验组戴模拟临床功能矫治器，引导大鼠下颌前伸，并在实验３ｄ，１，２，３和４周各处死５只大鼠，取下髁突，应用免疫组织化学方法探测β转化生长因子Ⅰ型受体（ＴβＲ－Ⅰ）在髁突中的含量及分布。结果：髁突各层软骨细胞均有Ⅰ型受体（ＴβＲ－Ⅰ）的分布，以成熟层阳性强度最高；功能矫形前伸下颌后，髁突软骨细胞各层ＴβＲ－Ⅰ的表达均增强。结论：机械刺激调节了ＴＧＦ－β的功能，从而促进骨的生长代谢     

转化生长因子β受体在体外培养的人牙乳头细胞中的表达

目的 探讨体外培养的人牙乳头细胞中转化生长因子β（ｔｒａｎｓｆｏｒｍｉｎｇ ｇｒｏｗｔｈ ｆａｃｔｏｒ－β,ＴＧＦ－β）受体的表达情况和外生的ＴＧＦ－β对人牙乳头细胞ＴＧＦ－β受体的表达情况和外源性培养、ＳＰ免疫组织化学及图像分析法,检测培养的人牙乳头细胞ＴＧＦ－β受体的表达情况和外源性ＴＧＦ－β对人牙乳头细胞ＴＧＦ－β受体的影响。结果 ＴＧＦ－βⅠ、Ⅱ型受体力强阳性表达,Ⅲ型受体为弱阳性表达。图     

TGF—β对人牙乳头间充质细胞增殖和细胞周期的影响

目的： 探讨ＴＧＦ－ β对人牙乳头间充质细胞增殖和细胞周期的影响。方法：采用ＭＴＴ法和流式细胞仪观察ＴＧＦ－β对人牙乳头细胞增殖和细胞周期的影响。结果：５０μｇ／Ｌ 组显著刺激人牙乳头细胞增殖（Ｐ＜ ０．０５），Ｇ１ ％ 显著降低，Ｓ％ 明显升高（Ｐ＜ ０ ．０１），反映细胞增殖活力的增殖指数ＰｒⅠ值（Ｓ＋ Ｇ２ Ｍ） 也明显增高（ Ｐ＜０ ．０１）。结论：ＴＧＦ－β可能通过促进人牙乳头细胞增殖参与牙胚发育和牙髓损伤修复。     

生长因子对人髁突软骨细胞DNA及胶原合成的影响

目的:研究胰岛素样生长因子(IGF-)、碱性成纤维样生长因子(bFGF)及转化生长因子-β1(TGF-β1)对人髁突软骨细胞增殖及代谢的调节作用。方法:采用体外细胞培养技术及同位素掺入法。结果:在0.4%新生小牛血清(NCS)条件下,bFGF对人髁突软骨细胞DNA合成有显著的促进作用,浓度在1ng/ml以上具显著性(P<0.05)。IGF-在10～100ng/ml呈剂量效应地促进3H-TdR的掺入,而TGF-β1无显著作用。bFGF在0.1～100ng/ml可显著促进人髁突软骨细胞3H-Proline掺入,最大效应剂量为10ng/ml(增加60%)。IGF-在10～100ng/ml能够明显促进细胞胶原合成,最大值在100ng/ml(增加98%)。TGF-β1在1～10ng/ml明显抑制3H-Proline掺入,最大抑制浓度为1ng/ml,抑制率约为24%。结论:生长因子可有效促进软骨细胞增殖及基质合成,为关节软骨损伤性疾患的治疗提供一种有效的途径     

下颌骨髁突软骨细胞传代过程中生物学特性变化

目的:观察体外培养及传代对人髁突软骨细胞生物学特性的影响。方法:采用体外细胞培养、免疫生化及分子生物学方法观察传代过程中人髁突软骨细胞形态、型胶原及蛋白多糖合成及、、型前胶原的mRNA水平变化。结果:传代过程中人髁突软骨细胞型胶原合成及其mRNA水平逐渐降低,至第7代已基本丧失。而、型胶原的mRNA水平随传代逐渐升高,同时伴有细胞蛋白多糖合成的减少,细胞壁形态由圆形或多角形转变为长梭形占优势。结论:体外培养及反复传代可影响软骨细胞的表型特征。髁突软骨细胞的反分化标准应从胶原、蛋白多糖合成及细胞形态等方面综合评价     

The effects of growth factors on DNA synthesis, proteoglycan synthesis and alkaline phosphatase activity in bovine dental pulp cells.

Platelet-derived growth factor (PDGF), insulin-like growth factor-I and -II (IGF-I and -II), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) stimulated [125I]-deoxyuridine incorporation about 13, 6.2-, 4.6-, 3.8-, 3.1- and 1.2-fold, respectively, above control values at a concentration of 50 ng/ml. Transforming growth factor-beta (TGF-beta) decreased incorporation about 30% at the same dose. aFGF, IGF-I, IGF-II, bFGF and TGF-beta increased [35S]-sulphate incorporation 231, 71, 64, 42 and 39%, respectively, in proliferating cells, while EGF, IGF-I, TGF-beta and PDGF decreased incorporation about 30%, and aFGF increased incorporation 80% in stationary-stage culture. TGF-beta, PDGF, aFGF and bFGF caused 65-40% inhibition of alkaline phosphatase activity in proliferating and stationary cultures. These findings suggest that the proliferation of pulp cells may be stimulated mainly by PDGF and IGF-I, and the production of extracellular matrix proteoglycan may be enhanced by aFGF, IGF-I and IGF-II. Furthermore, TGF-beta, PDGF, aFGF and bFGF may regulate the differentiation of pulp cells into odontoblasts.     

人胚颞颌关节软骨细胞培养及生物学特性研究

采用机械分离及胰蛋白酶和Ⅱ型胶原酶联合消化的方法，简便、快速地获得了大量成活率高的人ＴＭＪ软骨细胞。在ＤＭＥＭ培养基中进行原代和传代培养，并对原代培养软骨细胞进行了组化及免疫组化鉴定，相差显微镜、光镜及超微结构的观察。结果显示，光镜观察，原代培养细胞呈多角形，单层排列，电镜可见细胞内有丰富的粗面内质网及线粒体，细胞呈多极性，表面有突起。ＴＭＪ软骨细胞免疫组化Ⅱ型胶原染色阳性。甲苯胺蓝染色细胞质内有     

Mitogenic, chemotactic, and synthetic responses of rat periodontal ligament fibroblastic cells to polypeptide growth factors in vitro.

The mitogenic, chemotactic, and synthetic responses of rat periodontal ligament (PDL) fibroblastic cells to epidermal growth factor (EGF), transforming growth factor-beta (TGF-beta), recombinant human platelet-derived growth factor (rhPDGF)-AB, rhPDGF-BB, natural (n) PDGF-AB, and insulin-like growth factor-I (IGF-I) were examined in vitro using PDL cells obtained from the coagulum of healing tooth sockets. PDGFs and IGF-I have potent and comparable mitogenic effects on PDL fibroblastic cells. The maximum mitogenic effect of PDGFs was observed at the concentration of 10 ng/ml, whereas that of IGF-I was seen at concentrations higher than 100 ng/ml. In contrast, EGF induced moderate, and TGF-beta inhibitory mitogenic responses. The combination of rhPDGF-AB with either EGF or TGF-beta demonstrated comparable mitogenic potency, equivalent to the level of PDGF alone regardless of the mitogenic effect of other growth factors. The combination of rhPDGF-AB and IGF-I, however, showed a synergistic effect revealing the highest mitogenic effect among all individual growth factors as well as any combinations of the growth factors tested. Similarly, PDL fibroblastic cells demonstrated strong chemotactic responses to both IGF-I and PDGFs. The maximum effect was observed by IGF-I at concentrations higher than 10 ng/ml, followed by rhPDGF-BB at 0.1 ng/ml, rhPDGF-AB and nPDGF at concentrations ranging from 0.1 to 1 ng/ml. TGF-beta revealed no, and EGF slightly increased, chemotactic effects. IGF-I slightly enhanced the synthesis of total protein, whereas other factors had no significant effect. However, both rhPDGF-AB and TGF-beta stimulated collagen synthesis. On the other hand, IGF-I showed no effect on collagen synthesis, while EGF suppressed collagen synthesis. These findings suggest that rhPDGF-BB and IGF-I stimulate proliferation and chemotaxis of PDL fibroblastic cells. In addition, the combination of these growth factors further increases the mitogenic effect. rhPDGF-AB also stimulates collagen synthesis by PDL fibroblastic cells. Thus, rhPDGF-BB and IGF-I may have important roles in promotion of PDL healing, and consequently, may be useful for clinical application in periodontal regenerative procedures.     

Primary and secondary cartilages of the neonatal rat: the femoral head and the mandibular condyle

Primary and secondary cartilages differ in embryonic origin and in histological organization, and are generally considered to have a different mode of growth. However, few studies have directly compared the two types of cartilage of the same animal at the same age. Therefore, we analysed several histological and biochemical differences between secondary cartilage of the mandibular condyle and primary cartilage of the femoral head of 4-d-old rats. We evaluated the tissue organization, the level of DNA and glycosaminoglycan (GAG) synthesis, and the GAG and collagen content. The expression of collagen types I, II and III and of receptors for insulin-like growth factor (IGF)-I, fibroblast growth factor (FGF), and transforming growth factor (TGF)-beta were investigated by immunohistochemistry. The ex vivo DNA and GAG synthesis as well as the GAG content of femoral heads were much higher than that of mandibular condyles. Mandibular condyles expressed both collagen types I and II, while femoral heads expressed only type II collagen. In the mandibular condyles, receptors for IGF-I, FGF, and TGF-beta were observed mainly in the superficial layers, whereas they were found throughout the entire femoral head. In conclusion, major differences were found between both types of cartilage, which might be related to their specific functional demands.     

Effects of growth factors and glucosamine on porcine mandibular condylar cartilage cells and hyaline cartilage cells for tissue engineering applications

Temporomandibular joint (TMJ) condylar cartilage is a distinct cartilage that has both fibrocartilaginous and hyaline-like character, with a thin proliferative zone that separates the fibrocartilaginous fibrous zone at the surface from the hyaline-like mature and hypertrophic zones below. In this study, we compared the effects of insulin-like growth factor-I (IGF-I), basic fibroblast growth factor (bFGF), transforming growth factor beta1 (TGF-β1), and glucosamine sulphate on porcine TMJ condylar cartilage and ankle cartilage cells in monolayer culture. In general, TMJ condylar cartilage cells proliferated faster than ankle cartilage cells, while ankle cells produced significantly greater amounts of glycosaminoglycans (GAGs) and collagen than TMJ condylar cartilage cells. IGF-I and bFGF were potent stimulators of TMJ cell proliferation, while no signals statistically outperformed controls for ankle cell proliferation. IGF-I was the most effective signal for GAG production with ankle cells, and the most potent upregulator of collagen synthesis for both cell types. Glucosamine sulphate promoted cell proliferation and biosynthesis at specific concentrations and outperformed growth factors in certain instances. In conclusion, hyaline cartilage cells had lower cell numbers and superior biosynthesis compared to TMJ condylar cartilage cells, and we have found IGF-I at 100 ng/mL and glucosamine sulphate at 100 μg/mL to be the most effective signals for these cells under the prescribed conditions.