人胚胎长骨中骨形态发生蛋白的免疫组化研究

目的：探讨骨形态发生蛋白与人胚胎骨发生及发育的关系。方法：用免疫组化技术和图像分析系统研究了第9－24周人胚胎股骨中骨形态发生蛋白的分布规律及胎龄性变化。结果：第9周胚胎股骨为软骨锥形,雏形的软骨膜,软骨基质及其两端的软骨细胞骨形态发生蛋白呈阳性反应,第10－24周胚胎股骨中,骨形态发生蛋白阳性反应主要见于成骨细胞,骨膜内层细胞,新生骨细胞及骨基质中,破骨细胞及骨髓细胞骨形态发生蛋白也呈阳性反应,骺软骨为阴性,但其内的软骨管为阳性反应,图象分析测定表明：人胚胎股骨内成骨细胞及骨小梁的平均灰度值随胎龄增加而逐渐下降,同一股骨内不同部位的骨小梁和成骨细胞的平均灰度值有所不同,结论：骨形态发生蛋白与胚胎骨的发生及发育密切相关。     

血管内皮生长因子和缺氧诱导因子对软骨细胞凋亡的作用

背景：骨关节炎是一种关节疾病,主要影响软骨,随着软骨细胞胞外基质的变化,软骨细胞发生凋亡,血管内皮生长因子在促进血管内皮细胞分裂与增殖、诱导血管生成中起重要作用。缺氧诱导因子是一种在细胞环境中的转录因子,因氧含量而产生不同反应,血管内皮生长因子和缺氧诱导因子在抑制软骨细胞凋亡中的作用受到研究者的重视。 目的：阐述血管内皮生长因子和缺氧诱导因子及其他可能因素对软骨细胞凋亡的影响。 方法：分析、总结近年来软骨细胞凋亡的影响因素的相关文献,从骨关节炎进程中软骨细胞血管内皮生长因子表达变化,血管内皮生长因子和缺氧诱导因子对软骨细胞凋亡的调控等方面进行阐述。 结果与结论：血管内皮生长因子通过上调抑制细胞凋亡因子的表达促进软骨细胞存活,缺氧诱导因子能增加软骨细胞活性和细胞外基质合成,成为抑制骨细胞凋亡的重要靶点,血管内皮生长因子与缺氧诱导因子的相关性还有待进一步研究。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

血管内皮生长因子165/骨形态发生蛋白改善缺氧复氧状态下成骨细胞损伤

背景：研究发现,血管内皮生长因子165和骨形态发生蛋白两种因子在缺氧复氧过程中相互作用,通过调节细胞内信号通路的活化,参与骨细胞损伤的修复过程。目的：进一步探究血管内皮生长因子165/骨形态发生蛋白与缺氧复氧成骨细胞损伤的关系分析。方法：取成骨细胞,建立缺氧复氧损伤模型,建模前后Real-Time PCR法和免疫印迹法检测血管内皮生长因子165、骨形态发生蛋白2的mRNA及蛋白表达。分别给予建模后成骨细胞不同质量浓度(10,20,40ng/mL)血管内皮生长因子165或骨形态发生蛋白2处理12,24,36,48,72 h,CCK-8法检测细胞增殖,DAPI检测细胞凋亡。结果与结论：(1)与建模前相比,建模后成骨细胞中血管内皮生长因子165、骨形态发生蛋白2的mRNA和蛋白表达量显著降低(P <0.05);(2)成骨细胞增殖率随着血管内皮生长因子165质量浓度的升高而明显升高(P <0.05);成骨细胞凋亡率随着血管内皮生长因子165质量浓度的升高而明显降低(P <0.05);(3)成骨细胞增殖率随着骨形态发生蛋白2质量浓度的升高而明显升高(P <0.05);成骨...     

雌激素对成骨细胞血管内皮生长因子表达的影响

背景:在众多的血管生长因子中,血管内皮生长因子是最有力的血管生成因子,它具有促进血管内皮细胞分裂、增殖,细胞质钙化聚集并能诱导血管生成等作用,且对于成骨细胞、破骨细胞的增殖、分化及功能活性具有重要调节作用。目的:观察雌激素对成骨细胞上血管内皮生长因子表达的影响。方法:取新生48h大鼠颅盖骨,酶解消化得到成骨细胞,并进行体外培养。采用RT-PCR法测定10-10,10-8,10-6,10-4mol/L17β-雌二醇作用成骨细胞后血管内皮生长因子mRNA的表达。结果与结论:RT-PCR检测结果显示,血管内皮生长因子mRNA在原代成骨细胞内稳定表达;半定量分析证实,不同浓度17β-雌二醇组间血管内皮生长因子mRNA的表达量呈现一定规律,随着17β-雌二醇浓度的升高,血管内皮生长因子mRNA的表达量逐渐增加,10-6mol/L左右时,血管内皮生长因子mRNA的表达量最高,超过此剂量,血管内皮生长因子的表达下降。提示雌激素促进成骨细胞上血管内皮生长因子的表达上调,并存在剂量依赖性。     

人胎睾丸发生过程中VEGF的表达研究

目的:研究血管内皮生长因子(VEGF)在人胚胎睾丸组织发生过程中的表达特征,探讨其在睾丸发生中的作用。方法:采用HE染色观察睾丸发育,SP免疫组织化学法检测VEGF在不同胎龄睾丸组织中的表达变化。结果:人胚胎睾丸发育正常;VEGF在胎儿睾丸间质细胞、生殖细胞中均有不同程度表达,间质细胞以12～24周为表达高峰,生殖细胞以16～24周为表达高峰,24周后随着胎龄的增长均呈下降趋势(P<0.01)。结论:VEGF在人胎睾丸发生、发育过程中有不同程度的表达,表明其在睾丸发生过程中起着一定的作用。     

VEGF通过上调CCN2促HUVEC迁移和血管生成

目的 探讨血管内皮生长因子(VEGF)诱导的成骨细胞中结缔组织生长因子(CTGF/CCN2)对人脐静脉血管内皮细胞(HUVECs)的影响.方法 用Real time PCR法及ELISA法检测VEGF诱导成骨细胞(OSE)中CCN2含量;制备成骨细胞(OSE)上清液;将细胞分为control组、OSE组和VEGF-OSE组(n=3).用小干扰RNA (siRNA)转染法抑制成骨细胞中CCN2的表达;Transwell法检测内皮细胞迁移;Matrigel实验检测管样结构形成能力.结果 VEGF呈时间和剂量依赖性上调成骨细胞中CCN2 mRNA和蛋白的表达;CCN2可促进内皮细胞的迁移和管样结构形成(P＜0.05),当CCN2被siRNA基因沉默或者加入CCN2抗体后,CCN2对内皮细胞迁移和管样结构形成的促进作用均受到明显抑制(P＜0.05).结论 VEGF通过上调成骨细胞中CCN2的表达,促内皮细胞(HUVECs)的迁移和血管生成.     

神经生长因子可促进骨折愈合过程中血管内皮生长因子的表达

背景：骨折愈合过程十分复杂,需要多种细胞因子的参与。目前研究较多的细胞因子有骨形态发生蛋白、成纤维细胞生长因子、转化生长因子β、血管内皮细胞生长因子和胰岛素样生长因子,但神经生长因子在骨折愈合过程中对血管内皮细胞生长因子的作用尚不明确。 目的：观察神经生长因子对兔骨折愈合中血管内皮生长因子表达的影响。 方法：实验建立标准兔桡骨骨折模型,分别用神经生长因子、神经生长因子单克隆抗体和生理盐水进行干预,即应用神经生长因子组、拮抗神经生长因子组和对照组。 结果与结论：损伤后24,48 h和损伤后1,3,6,8周Western blot检测骨折端组织血管内皮生长因子蛋白的表达分析结果显示,3组各时间点血管内皮生长因子表达的关系为：应用神经生长因子组>对照组>拮抗神经生长因子组(P < 0.05)。结果证实,在骨折愈合过程当中,应用神经生长因子可以促进血管内皮生长因子表达。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

血管内皮生长因子家族及其受体在早期人胚胎肝发育过程中的作用

目的通过观察血管内皮生长因子A（VEGFA）、血管内皮生长因子C（VEGFC）及其受体flt-1、flk-1、flt-4在人胚胎肝发育过程中的表达，了解血管内皮细胞和造血细胞与人胚胎肝干细胞发育的关系。方法运用E3～12周胚胎，石蜡切片，免疫组织化学染色，光镜下观察人肝干细胞的发育、与血管内皮细胞和造血细胞的关系及其表达VEGFA、VEGFC等生长因子及其受体的情况。结果E3～5周人胚肝细胞具有幼稚细胞的形态特点，并星c—Met阳性反应。E10～12周，c—Met阳性的肝干细胞主要分布于汇管区周围。E4～6周胚，少数肝干细胞呈VEGFA免疫反应阳性，E7周以后阳性反应消失。VEGFC、flt-1、flk-1、flt-4阳性肝细胞在4～12周胚胎肝内都可检测到。E4～6周，靠近肝芽的横隔间充质中可见较多的血管内皮细胞，并正在形成血管，而在远离肝芽处的内皮细胞和毛细血管较少。E4、5周时，靠近原始肝的横隔间充质组织中血管内皮细胞呈VEGFA、VEGFC、flt-1、flk-1和flt-4阳性，肝内的血管内皮细胞为VEGFA、flt-1、flk-1、VEGFC和flt-4免疫反应阴性。E4～12周胚胎，造血细胞呈VEGFA和flt-4阳性反应，而VEGFC和flt-1、flk-1免疫反应阴性。结论肝干细胞的发育受肝外间充质血管产生的VEGFA、VEGFC的诱导和调节。造血细胞分泌的VEGFA可以通过旁分泌途径调节肝干细胞的发育，而造血细胞的发育依赖于肝干细胞产生的VEGFC。     

胎盘生长因子与妊娠

胎盘生长因子(PGF)是血管内皮生长因子(VEGF)家族的一个成员,主要在胎盘表达,但在病理情况下,PGF也在其他组织表达.在胎盘形成过程中,PGF可能直接或间接调节血管的形成、血管的通透性以及内皮细胞和滋养细胞成活.在妊高征、胎儿宫内发育迟缓(IUGR)、Down氏综合征和妊娠合并滋养细胞疾病中,PGF表达发生改变.     

血管内皮生长因子与胎儿生长受限的研究进展

血管内皮生长因子（VEGF）是一种特异的，强烈的血管内皮细胞促分裂因子和血管生成因子，在体内对于增加血管通透性及维持血管的正常状态和完整性也具有重要意义。它在胎盘组织有丰富的表达，通过自分泌和旁分泌机制对滋养叶细胞和内皮细胞的功能起特殊的调节作用。因而，VEGF在正常妊娠胎盘的形成和发育过程中起着重要作用，同时，研究发现其分泌异常与胎儿生长受限（FGR）的发生发展有密切关系。     

神经元型一氧化氮合酶和血管内皮生长因子在人胎早期小肠绒毛中的表达及意义

目的 探讨神经元型一氧化氮合酶(nNOS)血管内破生长因子(VEGF)在人胎早期小肠毛中的表达规律.方法 应用免疫组织化学SABC和PV法检测第2、3、4月龄段,nNOS和VEGF在14例人胎小肠绒千的表达.结果 第2月还胚龄时,nNOS在小肠绒千中呈阳性表达,VEGF在小肠绒毛上皮细胞呈阳性表达.第3-4月胎龄段,nNOS和VEGF在小肠绒毛中部分细胞均呈阳性表达.结论 nNOS和VEGF与人胎早期小肠绒毛的生长发育关系密切.     

胎儿顶骨发生过程中骨形态发生蛋白的分布

目的探讨骨形态发生蛋白(BMP)与人颅顶骨发生及发育的关系。方法用免疫组化技术和显微图像分析系统对第9～36w胎儿顶骨内BMP分布及含量进行了系统观察。结果第9w胎儿顶骨为一片间充质膜,呈BMP阳性反应;第10～36w胎儿顶骨内BMP阳性反应主要见于成骨细胞、新生骨细胞及骨膜中。图像分析表明:胎儿顶骨内BMP阳性的骨小梁及成骨细胞的平均灰度值(0～255,black～white)随胎龄增加而呈上升趋势。结论胎儿顶骨中BMP的含量随胎龄增加而下降,对顶骨的发生可能有启动作用。     

Immunolocalisation of vascular endothelial growth factor (VEGF) in human neonatal growth plate cartilage

Angiogenesis is essential for the replacement of cartilage by bone during growth and repair. In order to obtain a better understanding of the mechanisms regulating vascular invasion at sites of endochondral ossification we have investigated the expression of the endothelial cell-specific mitogen, vascular endothelial growth factor (VEGF), by chondrocytes in human neonatal growth plates. VEGF was absent from chondrocytes in the resting zone and only weakly expressed by occasional chondrocytes in the proliferating region. In the hypertrophic zone the number of chondrocytes stained and the intensity of staining for VEGF increased with chondrocyte hypertrophy, maximum expression of VEGF being observed in chondrocytes in the lower hypertrophic and mineralised regions of the cartilage. These observations provide the first demonstration of the presence of VEGF in situ in developing human bone and are consistent with in vitro observations demonstrating the upregulation of proangiogenic growth factor production with increasing chondrocyte hypertrophy. The presence of numerous small blood vessels and vascular structures in the subchondral region where VEGF expression was maximal indicates that VEGF produced by hypertrophic chondrocytes may play a key role in the regulation of vascular invasion of the growth plate.     

Endothelin-1 localization in bone cells and vascular endothelial cells in rat bone marrow

Endothelin-1 (ET-1) localization in bone cells and associated vascular endothelial cells in metaphyseal bone marrow of the rat femur was examined by a biotin-streptoavidin-horseradish peroxidase method in paraffin sections and by indirect immunogold techniques in post-embedded ultrathin sections. Mouse anti-ET-1 monoclonal antibody was used as the primary antibody. In metaphyseal bone marrow, intense immunostaining was observed over osteoclasts, osteoblasts, young osteocytes, and vascular endothelial cells. But bone and cartilage matrices and chondrocytes in the proliferating zone were negative for immunoreaction. At the subcellular level, specific immunogold labeling was localized along plasma membranes and in the cytoplasm including those of ruffled borders and clear zones of osteoclasts. Some colloidal gold particles were also detectable within pale vacuoles of osteoclasts. Immunoreactivity was also found along the plasma membranes, cisterns of rough-surfaced endoplasmic reticulum, mitochondria, and cytoplasmic matrices of osteoblasts, but was less intense than that of osteoclasts. In endothelial cells of blood capillaries in close proximity to bone cells, intense immunolabeling occurred over the cytoplasm. None of the cases examined showed accumulation of immunogold particles in the secretion granules of these cells. © 1993 Wiley-Liss, Inc.     

Cartilage canals in the chicken embryo are involved in the process of endochondral bone formation within the epiphyseal growth plate

A detailed study of so-called communicating cartilage canals, which penetrate deeply up into the lower hypertrophic zone of the epiphyseal growth plate in the embryonic chicken femur (E20), was carried out with the aim to clarify whether or not these canals are involved in the bone-forming process. In addition, we examined the manner in which cartilage canals are formed and compare the present data with our previous data. The canals were investigated by means of light microscopy, electron microscopy, immunohistochemistry (VEGF, VEGFR2/Flk1, type I collagen), and 3D reconstruction. Some communicating canals deeply penetrate into the upper hypertrophic zone where they terminate, showing electron-dense cells at their end. Subcellular characteristics of these cells are hardly detectable and we suppose that they undergo cell death. Other canals pass down deeper into the lower hypertrophic zone. The upper segment of these canals is composed of capillaries, mesenchymal cells, and macrophage-like cells. Precursors of osteoblasts are adjacent to the canals. The lower segment of communicating canals is composed of bone matrix or osteoid, which contains type I collagen fibrils and cells having the typical subcellular features of osteoblasts. No vessels are found in these segments. Immunohistochemistry shows that the matrix of the canals labels positively for type I collagen. In addition, staining with sirius red demonstrates that bone matrix is formed in these parts. We assume that the osteoblast-like cells of the lower segments of communicating canals originate either from mesenchymal cells or even from hypertrophic chondrocytes. Our immunohistochemical data also reveal that vascular endothelial growth factor (VEGF) and the corresponding receptor VEGFR2/Flk1 (VEGF receptor 2/Flk1) are localized in cartilage canals of the reserve zone, the proliferative zone, and the hypertrophic zone. The receptor is found in the endothelial cells of the vessels. Furthermore, VEGF is present in hypertrophic chondrocytes. The results of our study suggest that cartilage canals penetrate actively into the cartilage anlage and that bone is formed in the lower segments of the communicating canals where no vessels are detectable.     

In vivo leptin expression in cartilage and bone cells of growing rats and adult humans

The present investigation was carried out to analyse, immunohistochemically, in vivo leptin expression in cartilage and bone cells, the latter restricted to the elements of the osteogenic system (stromal cells, osteoblasts, osteocytes, bone lining cells). Observations were performed on the first lumbar vertebra, tibia and femur of four rats and on the humerus, femur and acromion of four patients. Histological sections of paraffin-embedded bone samples were immunostained using antibody to leptin. The results showed that, in growing rat bone, leptin is expressed in chondrocytes and stromal cells, but not in osteoblasts; bone lining cells were not found in the microscopic fields examined. In adult human bone, leptin is expressed in chondrocytes, stromal cells and bone lining cells; osteoblasts were not found in the microscopic fields examined. Osteocytes were found to be leptin positive only occasionally and focally in both rat and human bone. The in vivo findings reported show, for the first time, that leptin appears to be expressed only in the cells of the osteogenic lineage (stromal cells, bone lining cells, osteocytes) that, with respect to osteoblasts, are permanent and inactive, i.e. in those cells that according to our terminology constitute the bone basic cellular system (BBCS). Because the BBCS seems to be primarily involved in sensing and integrating mechanical strains and biochemical factors and then in triggering and driving bone formation and/or bone resorption, it appears that leptin seems to be mainly involved in modulating the initial phases of bone modelling and remodelling processes.     

The role of cartilage canals in endochondral and perichondral bone formation: are there similarities between these two processes?

We investigated the development of cartilage canals to clarify their function in the process of bone formation. Cartilage canals are tubes containing vessels that are found in the hyaline cartilage prior to the formation of a secondary ossification centre (SOC). Their exact role is still controversial and it is unclear whether they contribute to endochondral bone formation when an SOC appears. We examined the cartilage canals of the chicken femur in different developmental stages (E20, D2, 5, 7, 8, 10 and 13). To obtain a detailed picture of the cellular and molecular events within and around the canals the femur was investigated by means of three-dimensional reconstruction, light microscopy, electron microscopy, histochemistry and immunohistochemistry [vascular endothelial growth factor (VEGF), type I and II collagen]. An SOC was visible for the first time on the last embryonic day (E20). Cartilage canals were an extension of the vascularized perichondrium and its mesenchymal stem cell layers into the hyaline cartilage. The canals formed a complex network within the epiphysis and some of them penetrated into the SOC were they ended blind. The growth of the canals into the SOC was promoted by VEGF. As the development progressed the SOC increased in size and adjacent canals were incorporated into it. The canals contained chondroclasts, which opened the lacunae of hypertrophic chondrocytes, and this was followed by invasion of mesenchymal cells into the empty lacunae and formation of an osteoid layer. In older stages this layer mineralized and increased in thickness by addition of further cells. Outside the SOC cartilage canals are surrounded by osteoid, which is formed by the process of perichondral bone formation. We conclude that cartilage canals contribute to both perichondral and endochondral bone formation and that osteoblasts have the same origin in both processes.     

Development of the microcirculation of the secondary ossification center in rat humeral head

This work investigated the origin and development of microcirculation in the rat humeral head and the expression of vascular endothelial growth factor (VEGF) as a factor supporting the vascular growth and the development of the secondary ossification centers. Sixty rats aging 1, 3-4, 6-8, 11, and 21 days, 5 weeks, and 4 and 8 months were used. Samples of humeral head were collected for histology and immunohistochemistry for VEGF. Some animals were perfused with Mercox resin in order to obtain vascular corrosion casts (vcc) observed by scanning electron microscopy (SEM). No cartilage canals were present at birth. At 6 days postnatal, blood vessels coming from the perichondrium and the region near the capsule attachment invaded the cartilage; at 11 days postnatal, signs of calcification were present and within the third week some bone trabeculae were formed. Just before the vascular invasion of the epiphysis, a positive reaction for VEGF was localized in chondrocytes of the epiphyseal cartilage close to the capsule insertion. During the development and expansion of the secondary ossification center, VEGF expression was higher in chondrocytes but decreased when epiphysis was diffusely ossified. VEGF was expressed also by mesenchymal cells present in and around the fibrous tissue where the secondary ossification center will develop. SEM vcc confirmed that vessels penetrating into the epiphysis arose merely from the periosteal and the capsular networks, and vascular connections with the diaphyseal circulation were not evident. These observations demonstrated that VEGF production by chondrocytes begun some days after birth, supported the rapid vascular growth from the surrounding soft tissues, and was chronologically related to the development of the secondary ossification center in rat proximal humerus. Finally, the possible role of VEGF as mediator of angiogenesis and, at least indirectly, as a trigger factor also in the ossification and the bone remodeling of the secondary ossification centers has been discussed.     

Expression of vascular endothelial growth factor in normal, osteoarthritic and osteoporotic osteoblasts

To evaluate vascular endothelial growth factor (VEGF) mRNA expression and protein synthesis in primary human osteoblast cultures from healthy, osteoporotic and osteoarthritic subjects. Normal primary human osteoblast cultures were obtained from healthy subjects undergoing surgery for the reduction in traumatic fractures. Pathological osteoblasts were obtained from patients undergoing to total hip replacement for osteoporotic hip fracture or advanced osteoarthritis. VEGF mRNA expression and protein synthesis were evaluated in cultured cells, by semiquantitative real-time PCR and ELISA, respectively, both under basal conditions than after vitamin D₃ stimulation. Osteoarthritic osteoblasts showed a significantly higher VEGF expression compared to the normal and OP osteoblasts, both under basal conditions than in the presence of vitamin D₃, whereas no difference was found between osteoporotic and normal osteoblast. Vitamin D₃ significantly enhanced VEGF expression in normal and pathological osteoblasts. This preliminary study supports the hypothesis that VEGF is involved in the pathogenic mechanisms underlying the bone alterations typical of osteoarthritis and confirms the crucial role of vitamin D₃ supplementation in metabolic bone diseases.