软骨生长因子对兔软骨细胞作用的实验研究

目的：探讨软骨生长因子（CDGF）对体外培养兔软骨细胞的作用。研究了CDGF,胰岛素样生长因子－I（IGF－I）及软骨细胞生长代谢三者之间的关系。方法：体外单层培养兔软骨细胞,取生长良好的第二代细胞实验,分别或共同加入不同浓度CDGF和IGF－I,利用氯胺T法和MTTI地分别观察比较细胞培养液中羟脯氨酸与能量的合成及细胞的增殖情况,结果：CDGF与IGF－I均能剂量依赖性的促进细胞外基质中羟脯氨酸的合成以及细胞的增殖与能量合成;对两种作用的最佳刺激浓度,CDGF分别为16ng/ml和32ng/ml,IGF-I为30ng/ml;二者联合应用能明显增强对软骨细胞作用。结论：CDGF可以刺激软骨细胞的增殖与胶原合成,并与IGF－I有协同作用。     

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。     

Characterization of a heparin-binding growth factor from adenocarcinoma of the kidney

A polypeptide isolated from tissue extracts of renal adenocarcinoma was mitogenic for BALB/c 3T3 cells and human umbilical vein (HUV) cells in culture. It also demonstrated angiogenic ability using the chorioallantoic membrane bioassay. Using heparin-sepharose affinity chromatography the purified protein eluted with a NaCl concentration between 1.4 and 1.8 M and demonstrated a molecular weight of approximately 17,000 daltons based on SDS polyacrylamide gel electrophoresis. Half maximal stimulation of tritiated thymidine incorporation into BALB/c 3T3 cells was achieved by 1.6 ng./ml. of the heparin binding material. Western blot analysis using antibodies specific to basic fibroblast growth factor (bFGF) only or acidic FGF (aFGF) only demonstrated that the purified protein binds to the former and not the latter. The characteristics of this material, in effect the elution profile off heparin-Sepharose, the molecular weight, angiogenic activity and the results of western blot analysis, suggest that this growth factor is similar to the family of basic fibroblast growth factors.     

类肝素结合性表皮生长因子在人子宫内膜植入窗期的表达和意义

探讨类肝素结合性表皮生长因子 (HB-EGF)在人正常子宫内膜中的表达特点 ,评估其在子宫内膜的生长、分化及在植入窗期介导胚泡着床中的作用。采用免疫组织化学方法检测 5 6例正常子宫内膜 (1 6例增殖期 ,1 3例分泌早期 ,1 5例分泌晚期 ,1 2例植入窗期 )中 HB-EGF的表达及定位。结果 :HB-EGF在人各期子宫内膜均有表达 ,在植入窗期或分泌中期子宫内膜上皮细胞和间质细胞表达最强 ,并定位于腺上皮和腔上皮细胞表面 ,呈顶浆分泌状。结论 :HB-EGF存在于人各期子宫内膜 ,在植入窗期明显高表达可能参与介导胚泡着床 ,并与间质细胞的生长、分化有关 ,利于子宫内膜蜕膜化 ,为胚泡着床做准备。     

Leptin acts as a growth factor on the chondrocytes of skeletal growth centers.

Childhood obesity frequently is associated with an increase in height velocity and acceleration of epiphyseal growth plate maturation despite low levels of serum growth hormone (GH). In addition, obesity is associated with higher circulating levels of leptin, a 16-kDa protein that is secreted from the adipocytes. In this study, we evaluated the direct effect of leptin on the chondrocyte population of the skeletal growth centers in the mouse mandibular condyle, a model of endochondral ossification. We found that chondrocytes in the growth centers contain specific binding sites for leptin. Leptin, at a concentration of 0.5-1.0 microg/ml, stimulated in a dose-dependent manner the width of the chondroprogenitor zone (up to 64%), whereas higher concentrations had an inhibitory effect. Leptin induction of both proliferation and differentiation activities in the mandibular condyle was confirmed by our findings of an increase in bromodeoxyuridine (BrdU) incorporation into DNA and in (acidic) Alcian blue (AB) staining of the cartilaginous matrix. Leptin also increased the abundance of the insulin-like growth factor (IGF) I receptor and IGF-I receptor messenger RNA (mRNA) within the chondrocytes and the progenitor cell population. Our results indicate that leptin acts as a skeletal growth factor with a direct peripheral effect on skeletal growth centers. Some of its effects on the growing bone may be mediated by the IGF system via regulation of IGF-I receptor expression. We speculate that the high circulating levels of leptin in obese children might contribute to their growth.     

周期性应力下胰岛素样生长因子1受体对大鼠软骨细胞功能的影响

目的探讨周期性机械应力条件下胰岛素样生长因子1型受体(IGF1R)对大鼠软骨细胞增殖及细胞外基质合成的影响。 方法体外分离培养大鼠软骨细胞,随机分为4组：加压0 h组、加压0.5 h组、加压1 h组、加压2 h组,采用Western Blot法检测各组细胞IGF1R的表达及磷酸化水平,并在此基础上将同代无处理的软骨细胞在加压各组检测的同时给予同样检测即为静态组,应用Gel-Pro Analyzer软件进行半定量灰度分析比较各时间段静态和加压两两各组磷酸化IGF1R/总IGF1R水平差异。另取大鼠软骨细胞随机分为静态组、加压对照组、IGF1R阻断组、加压后IGF1R阻断组,IGF1R阻断采用顺式－3－[8－胺基－1－(2－苯基－喹啉－7－基)－咪唑并[1,5－a]吡嗪－3－基]－1－甲基－环丁醇(OSI－906)或者以IGF1R shRNA两种方式。阻断IGF1R 1 h后Western Blot法检测各组细胞磷酸化细胞外信号调节激酶1/2(ERK 1/2)表达及磷酸化水平;8 h后实时荧光定量PCR检测各组2型胶原(collagen Ⅱ)、蛋白聚糖(aggrecan)表达;3 d后采用直接细胞计数方式及细胞计数试剂盒(CCK-8)对软骨细胞增殖状况进行测定。应用SPSS 18.0软件进行相关统计学分析,两组间差异采用t检验比较。 结果磷酸化IGF1R/总IGF1R蛋白水平在加压0 h组与静态组之间差异无统计学意义(t＝0.255, P＝0.811),而在0.5 h组,1 h组,2 h组较相对应的静态组增高,差异具有统计学意义(t＝－5.881、－6.172、－10.518,P均小于0.05)。IGF1R被OSI-906或shRNA抑制后,加压处理的ERK 1/2磷酸化水平显著降低(OSI-906阻断后t＝3.074,shRNA阻断后t＝3.990,P均小于0.05),细胞外基质collagen Ⅱ(OSI-906阻断后t＝3.243, shRNA阻断后t＝3.621,均为P<0.05)、aggrecan(OSI-906阻断后t＝3.128,shRNA阻断后t＝3.608,P均小于0.05)基因表达水平显著降低,大鼠软骨细胞增殖能力减弱,差异均具有统计学意义(OSI-906阻断后t＝2.835、shRNA阻断后t＝3.467,均为P<0.05)。 结论周期性机械应力通过压力感受器IGF1R将机械信号转变为生物化学信号,激活ERK 1/2信号通路促进软骨细胞增殖和细胞外基质合成。     

重组人酸性成纤维细胞生长因子对人成骨细胞生长及增殖的影响

目的研究酸性成纤维细胞生长因子(acidfibroblastgrowthfactoraFGF)对成骨细胞生长及增殖的影响。方法取人胚胎颅盖骨骨内膜,利用组织块培养法进行成骨细胞原代培养,DMEM-F12培养液传代、扩增培养,以细胞形态学、细胞形成钙结节的能力及碱性磷酸酶活性鉴定成骨细胞。应用光镜、电镜、MTT法及流式细胞仪检测,分别从细胞形态、细胞生长及增殖特性分析不同浓度的aFGF对人成骨细胞生长和增殖的影响。结果10～100ng/mlaFGF可明显促进成骨细胞生长、增殖使S期细胞数增加,但高浓度aFGF(>1μg/ml)对成骨细胞生长具有抑制作用。结论aFGF对体外培养的人成骨细胞具有促生长和增殖作用,其有效浓度为10～100ng/ml。     

The effects of transforming growth factor β1, insulin-like growth factor 1 and leptin on the proliferation of fetal chondrocytes

One method to exogenously enhance the repair of articular cartilage is the local application of growth factors. This method is based on the chondrogenic effects of some agents and their potential ability to enhance cell migration. Human fetal chondrocytes were isolated and cultured. Their proliferation under the influence of different agents was microscopically evaluated. Fetal calf serum at 5% and 10% concentrations induced microscopically visible cell proliferation. Transforming growth factor beta one (5 and 10 ng/ml), insulin-like growth factor 1 (5 and 10 nmol/l) and leptin (1 and 2 ng/ml) accelerated proliferation of the cells towards the increasing gradient of the agents. Fibroblast growth factor beta (5 and 10 ng/ml), bone morphogenic protein two (10 ng/ml) and laminin (1 mg/ml) did not affect cell proliferation. This study suggests that different agents can play a role in the proliferation of fetal chondrocytes.     

Synergistic effect of transforming growth factor beta and fibroblast growth factor on DNA synthesis in chick growth plate chondrocytes

Transforming growth factor beta and fibroblast growth factor are mitogens for chick growth plate chondrocytes. TGF-beta stimulated a 3.5-fold increase, and FGF a 13.5-fold increase in the rate of thymidine incorporation after a 24 h exposure. TGF-beta and FGF were synergistic in chondrocytes, causing a 73-fold stimulation in thymidine incorporation compared with control. This synergistic response was not dependent upon the simultaneous presence of both mitogens. Sequential exposure of chondrocytes to TGF-beta and FGF in either order reproduced in large part the synergistic interaction observed when both growth factors were present simultaneously. The time required for induction of the subsequent synergistic response was brief and, in the case of TGF-beta, corresponded to the time required for [125I]TGF-beta receptor binding. EGF and PDGF were not mitogenic for chondrocytes, and neither of these factors enhanced the response of the cells to either TGF-beta or FGF. Finally, TGF-beta and FGF did not, either alone or in combination, elevate intracellular cAMP levels. These results emphasize the importance of examining growth factor effects in the context of other growth regulators. Furthermore, this specific and dramatic synergistic stimulation of thymidine incorporation may provide a useful tool in elucidating the mitogenic mechanism of the individual growth factors.     

Effects of hyaluronic acid and basic fibroblast growth factor on motility of chondrocytes and synovial cells in culture

The capacity of chondrogenic precursor cells to migrate and proliferate in an injured area is considered to be essential for cartilage repair. We examined cell motility of chondrocytes and synovial cells in monolayer culture and the chemokinetic effects of hyaluronic acid (HA) and basic fibroblast growth factors (bFGF) on these cells. The velocity of chondrocyte migration was accelerated by giving bFGF and simultaneously administering of both HA and bFGF, but it was not affected by HA alone. The velocity of synovial cell migration was increased by HA, but not by bFGF. HA had a chemokinetic effect on synovial cells and bFGF had the same effect on chondrocytes. Treatment with exogenous HA and bFGF may be of value for repairing articular cartilage injury by recruiting chondrogenic cells and promoting migration of chondrocytes in the cartilage tissue.     

Effects of hyaluronic acid and basic fibroblast growth factor on motility of chondrocytes and synovial cells in culture

The capacity of chondrogenic precursor cells to migrate and proliferate in an injured area is considered to be essential for cartilage repair. We examined cell motility of chondrocytes and synovial cells in monolayer culture and the chemokinetic effects of hyaluronic acid (HA) and basic fibroblast growth factors (bFGF) on these cells. The velocity of chondrocyte migration was accelerated by giving bFGF and simultaneously administering of both HA and bFGF, but it was not affected by HA alone. The velocity of synovial cell migration was increased by HA, but not by bFGF. HA had a chemokinetic effect on synovial cells and bFGF had the same effect on chondrocytes. Treatment with exogenous HA and bFGF may be of value for repairing articular cartilage injury by recruiting chondrogenic cells and promoting migration of chondrocytes in the cartilage tissue.     

Effects of hyaluronic acid and basic fibroblast growth factor on motility of chondrocytes and synovial cells in culture

The capacity of chondrogenic precursor cells to migrate and proliferate in an injured area is considered to be essential for cartilage repair. We examined cell motility of chondrocytes and synovial cells in monolayer culture and the chemokinetic effects of hyaluronic acid (HA) and basic fibroblast growth factors (bFGF) on these cells.

The velocity of chondrocyte migration was accelerated by giving bFGF and simultaneously administering of both HA and bFGF, but it was not affected by HA alone. The velocity of synovial cell migration was increased by HA, but not by bFGF. HA had a chemokinetic effect on synovial cells and bFGF had the same effect on chondrocytes. Treatment with exogenous HA and bFGF may be of value for repairing articular cartilage injury by recruiting chondrogenic cells and promoting migration of chondrocytes in the cartilage tissue.     

Effects of transforming growth factor-beta on aggrecanase production and proteoglycan degradation by human chondrocytes in vitro

OBJECTIVE: Aggrecan is degraded by Aggrecanases (ADAMTS-4 and -5) and MMPs, which cleave its core protein at different sites. Transforming growth factor (TGF)beta is known to stimulate matrix formation in cartilage, and ADAMTS-4 production in synoviocytes. The aim of this in-vitro study was to examine the effects of TGFbeta on aggrecanase production in human cartilage. DESIGN: Expression of ADAMTS-4 and -5 in chondrocyte cultures from normal or osteoarthritic cartilage was studied at mRNA level by RT-PCR. Aggrecanase activity was examined by western blot of aggrecanase-generated neoepitope NITEGE, and by measure of proteoglycan degradation in cartilage explants. RESULTS: TGFbeta strongly increased mRNA levels of ADAMTS-4, while ADAMTS-5 was expressed in a constitutive way in chondrocytes from normal and osteoathritic cartilage. TGFbeta also increased NITEGE levels and proteoglycan degradation. Addition of an aggrecanase inhibitor blocked the increase of NITEGE, and partially inhibited proteoglycan degradation. CONCLUSIONS: TGFbeta stimulates ADAMTS-4 expression and aggrecan degradation in cartilage. This catabolic action seems to be partially mediated by aggrecanases. It is, therefore, proposed that the role of TGFbeta in cartilage matrix turnover is not limited to anabolic and anti-catabolic actions, but also extends to selective degradation of matrix components such as aggrecan.     

Timing, route, and dose of administration of heparin-binding epidermal growth factor-like growth factor in protection against intestinal ischemia-reperfusion injury

Purpose

We have previously demonstrated that heparin-binding epidermal growth factor-like growth factor (HB-EGF) is an intestinal cytoprotective agent. The current study examined whether HB-EGF is effective as salvage therapy as well as prophylactic therapy for intestinal ischemia-reperfusion (I/R) injury, whether intravenous administration is as effective as intraluminal administration, and whether increased benefits are seen with increasing dose.

Methods

Total midgut I/R injury in rats was achieved by occlusion of a first-order branch of the superior mesenteric artery for 60 minutes, followed by reperfusion for 6 hours. Rats were treated with HB-EGF 5 minutes before ischemia, halfway through the ischemic event, or 5 minutes after ischemia. Route of administration was tested by administering HB-EGF either intraluminally or intravenously. Seven different doses of HB-EGF were tested.

Results

Heparin-binding, EGF-like growth factor protected the intestine from injury when administered before injury and was also effective when administered during ischemia or even after injury. Intraluminal administration of HB-EGF was superior to intravenous administration. Increasing doses of HB-EGF resulted in a greater cytoprotective effect.

Conclusion

These data demonstrate that HB-EGF acts as an effective intestinal cytoprotective agent when administered intraluminally not only before injury, but also during injury and, most importantly, even after intestinal injury has already occurred. These findings support a basis for the prophylactic use of intraluminal HB-EGF in high-risk patients, as well as for the administration of HB-EGF to salvage patients in whom an intestinal insult has already occurred.     

Effects of growth factors on cell proliferation and matrix synthesis of low-density, primary bovine chondrocytes cultured in collagen I gels.

Low cell density cell numbers and dedifferentiation are two major problems of human chondrocyte culture associated with articular cartilage repair. Bovine chondrocytes seeded at low density (3.5 x 10(4) cells/ml of gels) in three-dimensional collagen type I gels do proliferate and maintain their phenotype as shown by cell counts, morphology and matrix synthesis. The combination of three growth factors (3GFs; 10 ng/ml TGF-beta1 + 100 ng/ml IGF-I + 10 ng/ml b-FGF) added to serum-free culture medium in this culture system enhances the mitotic activity of bovine chondrocytes similar to 20% foetal calf serum (FCS). At day 21, cells proliferated by 41 fold in gels-FCS and 37 fold in gels-3GFs. Protein synthesis by gels-3GFs cultures was similar to 20% FCS when cultured for 3 weeks but much less proteoglycan was synthesized. The matrix deposition as observed by light and electron microscopy was quite different. More small diameter branching collagen fibrils and a denser matrix were presented in gels-FCS culture whilst loosely arranged larger diameter collagen fibrils were observed in gels-3GFs.     

Inhibition of heparin-binding epidermal growth factor-like growth factor increases albuminuria in puromycin aminonucleoside nephrosis

BACKGROUND: Our previous work in the acute puromycin aminonucleoside nephrosis (PAN) model has demonstrated up-regulation of heparin-binding epidermal growth factor-like growth factor (HB-EGF) mRNA and protein within glomerular epithelial cells (GECs) prior to the onset of proteinuria. METHODS: To determine whether increased HB-EGF expression in the acute PAN model contributes to the pathogenesis of proteinuria, a monoclonal antibody (DE10) was produced against recombinant human HB-EGF. RESULTS: The specificity of DE10 for human HB-EGF was confirmed by enzyme-linked immunosorbent assay, immunohistochemical staining, and flow cytometry of transfected cells expressing human and rat HB-EGF, and inhibition of cell proliferation. DE10 also reacted with cells transfected with rat HB-EGF cDNA. Administration of 0.5 mg affinity-purified DE10 to normal rats did not cause significant albuminuria compared with controls. Five days after the induction of the acute PAN model, albuminuria was significantly greater in animals treated with 0.5 mg DE10 than a control mAb (162.6 +/- 32.4 vs. 64.8 +/- 10.2 mg/day, respectively, P < 0.01). Rats treated with DE10 had an earlier onset of severe albuminuria, but no increase in maximal albuminuria at later time points. Electron microscopy showed marked podocyte effacement in both DE10-treated and control animals, but no obvious difference between groups. However, adhesion of the human GEC line 56/10 A1 to laminin and fibronectin, but not to collagens I or IV, was reduced by DE10. CONCLUSIONS: This study suggests that HB-EGF contributes to the integrity of the glomerular filtration barrier, particularly when the podocyte has been injured. Following podocyte injury, adhesion to laminin in the glomerular basement membrane by HB-EGF may be important in reducing albuminuria.