低氧促进大鼠心脏成纤维细胞DNA合成及Ⅰ、Ⅲ胶原mRNA表达

目的:研究低氧(2%氧)对成年Wistar大鼠心脏成纤维细胞DNA合成及Ⅰ、Ⅲ型胶原前a肽链表达的影响.方法:分离培养成年Wistar大鼠心脏成纤维细胞,采用液体闪烁计数方法检测心脏成纤维细胞的DNA合成速率,采用原位杂交技术检测Ⅰ、Ⅲ型胶原前α肽链mRNA的表达.结果:成年Wistar大鼠心脏成纤维细胞在低氧第6h、12 h时3H-TdR掺入量较常氧组显著增加,分别增加34%(P＜0.05)和36%(P＜0.01);低氧第4 h、8 h、12 h Ⅰ型胶原前α肽链mRNA表达显著高于常氧培养的细胞;低氧第2 h,Ⅲ型胶原前α肽链mRNA表达显著高于常氧培养的细胞.结论:低氧能够直接促进体外培养的成年Wistar大鼠心脏成纤维细胞DNA合成和Ⅰ、Ⅲ型胶原前α肽链表达,提示低氧对心脏成纤维细胞生长和胶原表达的直接调节可能是低氧性心肌纤维化的重要机制.     

粗纤维调节素对小鼠成纤维细胞Ⅰ型前胶原基因表达的影响

为了确定粗纤维调节素（Ｕｎ） 是否具有促进成纤维细胞Ⅰ型胶原合成的作用, 我们采用地高辛原位杂交和斑点杂交技术, 观察了Ｕｎ 对小鼠成纤维细胞（ＮＩＨ／３Ｔ３） Ⅰ型前胶原ｍ ＲＮＡ转录的影响; 结果发现Ｕｎ 对体外培养的３Ｔ３ 细胞Ⅰ型前胶原基因转录具有促进作用; 提示Ｕｎ 不仅作为一种细胞外基质而存在, 并且还有促进胶原合成的功能。     

皮肤成纤维细胞复合纤维修复前交叉韧带的初步研究

目的:本实验采用皮肤成纤维细胞修复原位冻融的前交叉韧带,以探索皮肤成纤维细胞作为构建组织工程前交叉韧带种子细胞的可行性.方法:体外分离培养兔皮肤成纤维细胞(SF),传代培养之后将细胞复合纤维生物蛋白胶,将细胞.纤维蛋白胶复合物植入原位冻融的前交叉韧带处.12周取材切片行HE染色及天狼猩红染色,使用偏振光显微镜观察.并使用图象分析软件对Ⅰ、Ⅲ型胶原含量进行半定量分析.结果:采用皮肤成纤维细胞复合纤维生物蛋白胶修复原位冻融的前交叉韧带的Ⅲ型胶原含量较单纯冻融的前交叉韧带明显减少,而较正常前交叉韧带则无明显统计学差异(P＞0.05).结论:皮肤成纤维细胞可作为构建组织工程前交又韧带的较为理想的种子细胞选择.     

碱性成纤维细胞生长因子对牙周膜细胞内核心蛋白多糖基因表达的影响

利用外源性碱性成纤维细胞生长因子(Basic Fibroblast Growth Factor,bFGF)刺激体外培养的人正常牙周膜细胞.采用逆转录-聚合酶链式反应(RT-PCR)检测细胞内decorin的基因表达的变化,研究bFGF对体外培养的人牙周膜细胞内核心蛋白多糖(decorin)的作用,进一步探讨bFGF抑制Ⅰ型胶原的作用机制.发现bFGF刺激牙周膜细胞后能促进牙周膜细胞的增殖,bFGF抑制decorin的合成是bFGF促进牙周膜细胞增殖的重要调节因素之一.     

TGF-β1在应力介导的牙周膜成纤维细胞增殖中的作用及其机制

目的:探讨周期性张应力作用下人牙周膜成纤维细胞(HPDLF)转化生长因子β1(TGF-β1)对其细胞增殖的作用,及对其I型胶原基因表达的作用和影响.方法:在成功构建人牙周膜成纤维细胞体外培养力学刺激模型的基础上,利用多通道细胞牵张应力加载系统,对细胞分别施加2、6、12与24 h的周期性张应力,以不加力组为对照组,观察各组细胞形态变化,利用细胞计数试剂8检测细胞增殖活性,并利用ELISA试剂盒检测加力后各组TGF-β1的表达,并对加力12h组加入TGF-β1抑制剂SB431542,利用RT-PCR检测技术检测对I型胶原基因表达的影响.结果:与对照组比较,加力2h细胞增殖稍降低,6h增殖活性增强,12h达到峰值,24h增殖活性明显受到抑制;TGF-β1的表达与细胞增殖成正相关;TGF-β1受到抑制后细胞增殖受到影响,I型胶原的表达也受到影响.结论:人牙周膜成纤维细胞的增殖在一定时间的周期性张应力作用下先增加然后再抑制,其中TGF-β1参与细胞增殖,并且TGF-β1对人牙周膜成纤维细胞I型胶原的表达起促进作用.     

缺氧和DDPH对人胚肺成纤维细胞前胶原mRNA表达的影响

用核酸原位杂交和图像分析等方法,观察直接缺氧（Ｈ）和缺氧猪肺动脉内皮细胞条件培养液（ＨＥＣＣＭ）对人胚肺成纤维细胞（ＫＭＢ１７）的前胶原ｐｒｏα１（Ⅰ）,ｐｒｏα１（Ⅲ）ｍＲＮＡ表达和抗高血压药１－（２,６－二甲基苯氧基）－２－（３,４－二甲氧基苯乙氨基）丙烷盐酸盐（ＤＤＰＨ）对此过程的影响。结果发现,Ｈ和ＨＥＣＣＭ均可使ＫＭＢ１７的两型前胶原ｍＲＮＡ表达量增高,明显高于对照组（Ｐ＜０．０１）。ＤＤＰＨ对ＨＥＣＣＭ组细胞的Ⅰ,Ⅲ两型前胶原ｍＲＮＡ表达增高均有显著的抑制作用（抑制率分别为－４３．９７％和－５６．２２％）,而对Ｈ组仅抑制ｐｒｏα１（Ⅰ）前胶原ｍＲＮＡ的过量表达（－５３．５８％）。提示缺氧可直接或通过肺动脉内皮细胞的介导,促进人胚肺成纤维细胞的Ⅰ、Ⅲ两型前胶原ｍＲＮＡ表达,ＤＤＰＨ在基因转录水平上对此过程有抑制作用     

骨形态发生蛋白9定向诱导多潜能干细胞成骨分化

为确认和评价骨形态发生蛋白9(bone morphogenetic protein 9, BMP9)定向诱导多潜能干细胞成骨分化的能力,以鼠间充质干细胞C3H10、小鼠胚胎成纤维细胞(mouse embryonic fibroblasts,MEFs)和骨髓基质细胞(bone marrow stromal cell, BMSC)三种多潜能干细胞为目标细胞,用重组腺病毒的方法将BMP9导入细胞,通过荧光素酶报告基因实验、碱性磷酸酶(alkaline phosphatase,ALP)定量测定、钙盐沉积实验、real time PCR、动物实验和组织化学染色等方法,观察BMP9对于多潜能干细胞成骨分化的定向诱导作用．结果提示,BMP9能诱导C3H10、MEFs和BMSC细胞ALP的表达,且具有剂量依赖性．BMP9在体外能够促进C3H10细胞和MEFs细胞的钙盐沉积．经BMP9刺激后,C3H10细胞成骨相关基因ALP、Runx2、骨桥素(osteopontin, OPN)和骨钙素(osteocalcin, OC)的mRNA水平均增加．荧光素酶报告基因实验证实,BMP9可以活化Smad和成骨关键基因Runx2．动物实验和组织化学染色检查显示,BMP9可以诱导C3H10细胞在裸鼠皮下异位成骨,因此,BMP9具有定向诱导多潜能干细胞成骨分化的能力．     

回转模拟失重对心肌成纤维细胞Ⅰ型胶原代谢的影响

为探讨模拟失重对心肌成纤维细胞Ⅰ型胶原代谢的影响 ,本研究采用回转器模拟失重效应 ,通过免疫细胞化学和反转录聚合酶链式反应 (RT PCR)研究了回转模拟失重对原代培养的新生大鼠心肌成纤维细胞Ⅰ型胶原蛋白及mRNA表达 ,以及胶原降解抑制物———金属蛋白酶组织抑制因子 (Tissueinhibitorofmetallopro teinase ,TIMP)mRNA表达的影响。免疫细胞化学染色显示回转组Ⅰ型胶原蛋白沉积增加 ;RT PCR分析显示Ⅰ型胶原α1链 (TypeⅠcollagenα1chain ,ColⅠA1)mRNA表达没有明显变化 ,TIMP 1、TIMP 2及TIMP 3的mRNA表达均增强。提示在回转模拟失重条件下 ,心肌成纤维细胞Ⅰ型胶原蛋白沉积增加 ,作为胶原降解抑制物的TIMP可能是造成胶原沉积的原因     

BMP2诱导C3H10细胞成骨分化的TGF-βⅠ型受体的体外分析

筛选和分析与BMP2诱导间充质干细胞C3H10成骨分化有关的TGF-βⅠ型受体.利用显性负性突变型TGF-βⅠ型受体竞争抑制配体功能的特性,运用碱性磷酸酶定量测定、Real time PCR等方法,初步筛选出可能与BMP2诱导间充质干细胞C3H10成骨分化有关的的TGF-βⅠ型受体,随后运用RNA干扰的方法抑制相应TGF-βⅠ型受体的表达,进一步证实相关TGF-βⅠ型受体与BMP2发挥诱导成骨活性的关系.结果证实,显性负性突变的ALK3和ALK6能够抑制BMP2诱导的C3H10细胞成骨分化;RNA干扰抑制ALK3或(和)ALK6表达后,BMP2诱导C3H10细胞向成骨分化的趋势受到抑制.因此,ALK3和ALK6是与BMP2诱导C3H10细胞成骨分化有关的TGF-βⅠ型受体.     

恒牙牙根不同发育阶段牙髓中基质金属蛋白酶-8和I型胶原的表达

目的探讨Ⅰ型胶原及MMP-8在牙齿发育不同阶段的表达及作用。方法将人牙根不同发育阶段的牙髓分成三组,即牙根开始发育组、发育中组、根尖孔闭合组。采用SABC法对标本石蜡切片进行Ⅰ型胶原及MMP-8的免疫组化染色。结果Ⅰ型胶原与MMP-8均存在于牙髓中的成牙本质细胞、成纤维细胞、血管壁及细胞外基质中。牙根刚开始发育时染色最强,根尖孔闭合时染色最弱。Ⅰ型胶原与MMP-8的表达呈正相关关系。结论①牙本质和前期牙本质中Ⅰ型胶原主要是由成牙本质细胞分泌的,且随着牙齿发育,分泌胶原能力减弱;②MMP-8参与牙齿成熟过程;③MMP-8参与牙齿发育中Ⅰ型胶原的降解,有维持牙髓中胶原稳定的作用。     

Phosphatidylinositol-dependent bond between alkaline phosphatase and collagen fibers in the periodontal ligament of rat molars

Alkaline phosphatase (ALP) is anchored to the outer leaflet of the lipid bilayer via phosphatidylinositol (PI) and ALP activity has been localized in the plasma membrane of numerous tissues. In the periodontal ligament ALP activity is found in the collagen fibers in addition to the plasma membrane of the osteoblasts and fibroblasts. In this study, we examined the distribution of ALP activity in the periodontal ligament of rat molars and also examined whether the bond between ALP and collagen fibers is dependent on PI by using phosphatidylinositol-specific phospholipase C (PI-PLC). ALP activity was distributed in the periodontal ligament. The activity mirrored the distribution of collagen fibers in the periodontal ligament. Cytochemical analysis also demonstrated that ALP activity was located not only in the plasma membrane of fibroblasts, but also in the collagen fiber bundles and fibrils in the periodontal ligament. After treatment with PI-PLC, the loss of ALP activity in the periodontal ligament was observed histochemically, and the loss of ALP activity in the fibroblasts as well as in the collagen fiber bundles and fibrils was observed cytochemically. These results strongly indicate that the bond between ALP and the collagen fibers is also dependent on PI.     

A simple cell motility assay demonstrates differential motility of human periodontal ligament fibroblasts, gingival fibroblasts, and pre-osteoblasts

During periodontal regeneration, multiple cell types can invade the wound site, thereby leading to repair. Cell motility requires interactions mediated by integrin receptors for the extracellular matrix (ECM), which might be useful in guiding specific cell populations into the periodontal defect. Our data demonstrate that fibroblasts exhibit differential motility when grown on ECM proteins. Specifically, gingival fibroblasts are twice as motile as periodontal ligament fibroblasts, whereas osteoblasts are essentially non-motile. Collagens promote the greatest motility of gingival fibroblasts in the following order: collagen III>collagen V>collagen I. Differences in motility do not correlate with cell proliferation or integrin expression. Osteoblasts display greater attachment to collagens than does either fibroblast population, but lower motility. Gingival fibroblast motility on collagen I is generally mediated by α2 integrins, whereas motility on collagen III involves α1 integrins. Other integrins (α10 or α11) may also contribute to gingival fibroblast motility. Thus, ECM proteins do indeed differentially promote the cell motility of periodontal cells. Because of their greater motility, gingival fibroblasts have more of a potential to invade periodontal wound sites and to contribute to regeneration. This finding may explain the formation of disorganized connective tissue masses rather than the occurrence of the true regeneration of the periodontium. This research was supported by the Louisiana Board of Regents through the Millennium Trust Health Excellence Fund, HEF-(2000-05)-04.     

Activity of 25-Hydroxylase in Human Gingival Fibroblasts and Periodontal Ligament Cells

Background

We previously demonstrated that 25-hydroxyvitamin D₃ concentrations in gingival crevicular fluid are 300 times higher than those in the plasma of patients with aggressive periodontitis. Here we explored whether 25-hydroxyvitamin D₃ can be synthesized by periodontal soft tissue cells. We also investigated which of the two main kinds of hydroxylases, CYP27A1 and CYP2R1, is the key 25-hydroxylase in periodontal soft tissue cells.

Methodology/Principal Findings

Primary cultures of human gingival fibroblasts and periodontal ligament cells from 5 individual donors were established. CYP27A1 mRNA, CYP2R1 mRNA and CYP27A1 protein were detected in human gingival fibroblasts and periodontal ligament cells, whereas CYP2R1 protein was not. After incubation with the 25-hydroxylase substrate vitamin D₃, human gingival fibroblasts and periodontal ligament cells generated detectable 25-hydroxyvitamin D₃ that resulted in the production of 1α,25-dihydroxyvitamin D₃. Specific knockdown of CYP27A1 in human gingival fibroblasts and periodontal ligament cells using siRNA resulted in a significant reduction in both 25-hydroxyvitamin D₃ and 1α,25-dihydroxyvitamin D₃ production. Knockdown of CYP2R1 did not significantly influence 25-hydroxyvitamin D₃ synthesis. Sodium butyrate did not influence significantly CYP27A1 mRNA expression; however, interleukin-1β and Porphyromonas gingivalis lipopolysaccharide strongly induced CYP27A1 mRNA expression in human gingival fibroblasts and periodontal ligament cells.

Conclusions

The activity of 25-hydroxylase was verified in human gingival fibroblasts and periodontal ligament cells, and CYP27A1 was identified as the key 25-hydroxylase in these cells.     

Mechanical force inhibits osteoclastogenic potential of human periodontal ligament fibroblasts through OPG production and ERK‐mediated signaling

Periodontal ligament and gingival fibroblasts play important roles in bone remodeling. Periodontal ligament fibroblasts stimulate bone remodeling while gingival fibroblasts protect abnormal bone resorption. However, few studies had examined the differences in stimulation of osteoclast formation between the two fibroblast populations. The precise effect of mechanical forces on osteoclastogenesis of these populations is also unknown. This study revealed that more osteoclast‐like cells were induced in the co‐cultures of bone marrow cells with periodontal ligament than gingival fibroblasts, and this was considerably increased when anti‐osteoprotegerin (OPG) antibody was added to the co‐cultures. mRNA levels of receptor activator of nuclear factor‐kappaB ligand (RANKL) were increased in both populations when they were cultured with dexamethasone and vitamin D₃. Centrifugal forces inhibited osteoclastogenesis of both populations, and this was likely related to the force‐induced OPG up‐regulation. Inhibition of extracellular signal‐regulated kinase (ERK) signaling by a pharmacological inhibitor (10 µM PD98059) or by siERK transfection suppressed the force‐induced OPG up‐regulation along with the augmentation of osteoclast‐like cells that were decreased by the force. These results suggest that periodontal ligament fibroblasts are naturally better at osteoclast induction than gingival fibroblasts, and that centrifugal force inhibited osteoclastogenesis of the periodontal fibroblasts through OPG production and ERK activation. J. Cell. Biochem. 106: 1010–1019, 2009. © 2009 Wiley‐Liss, Inc.     

Regeneration therapy for oral disease

The aim of this paper is to provide a review of the current understanding of the mechanisms, cell and factors required for regeneration and restoration of periodontal tissue around natural teeth. Periodontal regeneration is a complex multifactorial process involving cell populations: periodontal ligament cells, bone cells, gingival fibroblasts and epithelial cells. This paper describes bone graft, guided tissue regeneration and enamel matrix derivative with the application of growth factors.     

Isolation and characterization of cultured human periodental ligament fibroblast-specific cDNAs

The molecular mechanisms that control the function of periodontal ligament (PDL) fibroblasts remain unclear. We speculated that the character of differentiating PDL fibroblasts is defined by the altered expansion of specific genes not found in neighboring gingival fibroblasts in the periodontium. To expand this set, subtractive hybridization was applied between cultured human PDL and gingival fibroblasts to identify genes differentially expressed in PDL. Consequently five candidate clones, PDLs (periodontal ligament specific) 5, -17, -22, -25, and -31 were identified and characterized by homology search, Northern analysis, and in situ hybridization. Although the mRNAs of these clones were expressed by bone marrow cells and rarely by gingival fibroblasts, the highest expression was detected in the PDL cells, which were uniformly distributed throughout the whole PDL. Amongst the five candidate clones, we focused on PDLs17, because it is a hypothetical protein whose biological function has not been reported yet in the database. Polyclonal antiserum raised against PDLs17 peptide was made, and stained the PDL fibroblasts, osteoblast-like cells and stromal cells in the bone marrow, but not gingival fibroblasts. The results suggest that clones, PDLs5, -17, -22, -25, and -31 may be used as PDL fibroblast-specific markers, and that PDLs17 could act as an important factor in the differentiation process of PDL fibroblasts.     

Biochemical comparison of fibroblast populations from different periodontal tissues: characterization of matrix protein and collagenolytic enzyme synthesis

To compare the expression of extracellular matrix components by fibroblasts from different periodontal tissues, rat molar periodontal ligament fibroblasts (RPL) and rat gingival fibroblasts (RGF) were isolated and cultured from individual animals. Pulse-chase experiments using [35S]methionine as a precursor revealed that confluent populations of early passage cells of both cell types synthesized similar amounts of collagen, fibronectin, and SPARC/osteonectin. Qualitative and quantitative differences were apparent in the relative proportions of type III collagen, in the rates of procollagen processing, and in the synthesis of a small number of unidentified proteins observed by sodium dodecyl sulphate--polyacrylamide gel electrophoresis. Collagen constituted 24-26% of the radiolabelled proteins secreted by both cell types, type I being the predominant collagen, with lower amounts of type III (3-8% RGF, 8-18% RPL) and type V (approximately 1%) collagens. Procollagen processing in the culture medium of RPL cells was more rapid than for RGF cells, but was increased in multilayered cultures of both RPL and RGF. In multilayered cultures, collagen TCA fragments, indicative of tissue collagenase activity, were also identified. Active and latent tissue collagenases and a latent form of a novel collagenolytic enzyme (matrix metalloendoproteinase-V) that cleaves native TCA fragments were demonstrated in these cultures. Addition of either concanavalin A (10(-6) M) or retinoic acid (10(-5) M) to the culture medium stimulated the secretion of the latent collagenolytic enzymes. Collagenase inhibitor was also synthesized by both RGF and RPL cells. SPARC/osteonectin, a 40-kilodalton glycoprotein, represented 0.5-1.0% of the secreted radiolabelled proteins of both cell types.     

Different effects of 25-kDa amelogenin on the proliferation, attachment and migration of various periodontal cells

Previous studies have assumed that amelogenin is responsible for the therapeutic effect of the enamel matrix derivative (EMD) in periodontal tissue healing and regeneration. However, it is difficult to confirm this hypothesis because both the EMD and the amelogenins are complex mixtures of multiple proteins. Further adding to the difficulties is the fact that periodontal tissue regeneration involves various types of cells and a sequence of associated cellular events including the attachment, migration and proliferation of various cells. In this study, we investigated the potential effect of a 25-kDa recombinant porcine amelogenin (rPAm) on primarily cultured periodontal ligament fibroblasts (PDLF), gingival fibroblasts (GF) and gingival epithelial cells (GEC). The cells were treated with 25-kDa recombinant porcine amelogenin at a concentration of 10 μg/mL. We found that rPAm significantly promoted the proliferation and migration of PDLF, but not their adhesion. Similarly, the proliferation and adhesion of GF were significantly enhanced by treatment with rPAm, while migration was greatly inhibited. Interestingly, this recombinant protein inhibited the growth rate, cell adhesion and migration of GEC. These data suggest that rPAm may play an essential role in periodontal regeneration through the activation of periodontal fibroblasts and inhibition of the cellular behaviors of gingival epithelial cells.     

Extracellular matrix regulates induction of alkaline phosphatase expression by ascorbic acid in human fibroblasts

During wound healing and inflammation, fibroblasts express elevated alkaline phosphatase (ALP), but are not in contact with collagen fibrils in the fibronectin (FN)-rich granulation tissue. We hypothesized that the extracellular matrix (ECM) environment might influence the induction of ALP in fibroblasts. Here we tested this hypothesis by studying the ALP-inductive response of normal human gingival fibroblasts to ascorbic acid (AsA). AsA induced ALP activity and protein in cells in conventional monolayer culture. This induction was inhibited by blocking-antibodies to the FN receptor alpha 5 beta 1 integrin and by the proline analog 3,4-dehydroproline (DHP). DHP prevented cells from arranging FN fibrils into a pericellular network and reduced the activity of cell spreading on FN. Plating of cells on FN facilitated the up-regulation by AsA of ALP expression, but did not substitute for AsA. In contrast, AsA did not cause ALP induction in cells cultured on and in polymerized type I collagen gels. Collagen fibrils inhibited the up-regulation by AsA of ALP expression in cells plated on FN. These results indicate that the ECM regulates the induction of ALP expression by AsA in fibroblasts: FN enables them to express ALP in response to AsA through interaction with integrin alpha 5 beta 1, whereas type I collagen fibrils cause the suppression of ALP expression and overcome FN.