脂多糖对大鼠牙髓细胞ALP、BSP、DSPP表达的影响

目的:研究脂多糖(lipopolysaccharide,LPS)对大鼠牙髓细胞牙本质涎磷蛋白(dentin sialophosphoprotein,DSPP)、骨涎蛋白(bone sialoprotein,BSP)及碱性磷酸酶(alkaline phosphatase,ALP)表达的影响。方法:采用组织块法获得大鼠牙髓细胞,体外常规培养并进行鉴定,以含0.1、1、10、100和10000 ng/mL牙龈卟啉单胞菌(Porphyromonas gingivalis,P.g)的LPS作用牙髓细胞1、3、5 d,用实时定量PCR检测DSPP、ALP、BSP mRNA表达的变化,采用SPSS17.0软件包对数据进行统计学分析。结果:镜下贴壁后的细胞形态多样,多呈成纤维样细胞形态,还有部分多角形细胞,胞质突起。实时定量PCR结果显示,与对照组相比,1、10 ng/mL LPS组大鼠牙髓细胞DSPP、ALP、BSP的mRNA表达增高,100、10000 ng/mL LPS组DSPP、ALP、BSP的mRNA表达均降低;在1、3、5 d时,1、10、100和10000 ng/mL LPS组mRNA表达逐渐减少。0.1 ng/mL LPS对mRNA的表达无显著影响。3种因子呈现相似的表达变化趋势。结论:低剂量P.g LPS能促进牙髓细胞ALP、BSP、DSPP的表达,高剂量时则抑制ALP、BSP、DSPP的表达;随着培养时间的延长,促进作用逐渐减弱,抑制作用逐渐增强。     

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.     

永生化成牙本质细胞表达牙本质基质蛋白的实验研究

目的　探讨永生化人成牙本质细胞样细胞系hTERT-hOd-l表达牙本质基质蛋白的情况。方法　矿化液培养hTERT-hOd-l细胞5周,检测骨钙素(OC)分泌量和碱性磷酸酶(ALP)活性。采用免疫组织化学、RT-PCR和原位杂交方法检测Ⅰ型胶原、骨涎蛋白(BSP)、牙本质基质蛋白1 (DMP1)以及成牙本质细胞标志物牙本质涎磷蛋白(DSPP) 和牙本质涎蛋白(DSP)在细胞中的表达。结果　在矿化液诱导下,hTERT-hOd-l细胞ALP活性和OC分泌量升高。 hTERT-hOd-l细胞在mRNA水平上表达BSP、DMP1和DSPP,在蛋白质水平上表达DSP和Ⅰ型胶原。结论　hTERT- hOd-l细胞在体外表达牙本质基质蛋白,具有矿化的潜能。     

Expression of mineralization markers in dental pulp cells

There is an increasing interest in the utility of dental pulp stem cells (DPSCs) for dentin regeneration. The mechanisms involved in DPSC differentiation remain poorly understood. The purpose of the study was to investigate the mineralization capacity of human dental pulp cells (DPCs) and identify potential markers for odontoblast differentiation. The isolated DPCs expressed mesenchymal stem-cell markers as shown by flow cytometry and could differentiate in vitro into odontogenic, adipogenic, and chondrogenic lineages. Alkaline phosphatase activity of DPCs elevated over time, with significant upregulation on day 21 in odontogenic induction. Quantitative RT-PCR revealed that osteocalcin, dentin sialophosphoprotein (DSPP), and matrix extracellular phosphoglycoprotein (MEPE) expression also increased time dependently in the induction cultures. In conclusion, we isolated DPCs with stem cell characteristics. MEPE and DSPP showed a similar regulatory pattern of DPCs mineralization. MEPE along with DSPP may be potential odontogenetic differentiation markers.     

Periodontal ligament cells from insulin-dependent diabetics exhibit altered alkaline phosphatase activity in response to growth factors.

BACKGROUND: Insulin-dependent or Type 1 diabetes mellitus (IDDM) has been associated with an increased severity of periodontal disease. Because periodontal ligament (PDL) cells play a significant role in maintenance and regeneration of mineralized tissue, the success of procedures, such as guided tissue regeneration, is directly related to the ability of these cells to augment mineralized tissue. The objective of this study was to examine the ability of PDL cells from long-standing IDDM patients to form mineralized tissue and to determine whether these cells would exhibit altered responses to exogenously added growth factors. METHODS: PDL cells were isolated from 4 patients with IDDM treated with insulin for at least 5 years and from systemically healthy donors. The cell isolates were tested for their ability to form mineralized nodules in vitro and to express alterations in alkaline phosphatase activity in response to exogenously added growth factors (transforming growth factor-beta (TGF-beta), platelet-derived growth factor-BB (PDGF-BB), and insulin-like growth factor-1 (IGF-1). Alkaline phosphatase activity was determined spectrophotometrically. RESULTS: Although all PDL cell isolates formed mineralized nodules in vitro, PDL cells from diabetics formed mineralized nodules more slowly than did the controls. Alkaline phosphatase activity was not altered by exposure of diabetic PDL cells to TGF-beta for 9 days. In contrast, non-diabetic isolates exhibited increased levels of activity with increasing concentrations, from 0.5 to 1.0 ng/ml. Alkaline phosphatase activity was significantly higher in non-diabetic, but not in diabetic, cell isolates exposed to TGF-beta at 1.0 ng/ml, when compared to non-treated controls. Diabetic cell isolates exhibited significantly lower alkaline phosphatase activity than the non-diabetic isolates when exposed to either TGF-beta, PDGF-BB, IGF-1 or a combination of PDGF-BB and IGF-1. CONCLUSIONS: These results suggest that the populations of PDL cells in insulin-dependent diabetics may be altered in their ability to form mineralized tissue and to respond to growth factors, functions affecting the maintenance and regeneration of the periodontium.     

脂多糖对诱导分化的牙髓细胞形成矿化基质的影响

目的 研究脂多糖对诱导分化的牙髓细胞牙本质涎磷蛋白(dentin sialophosphoprotein,DSPP)、骨钙素基因表达及碱性磷酸酶(alkaline phosphatase,ALP)活性的影响. 方法 以脂多糖作用于诱导分化的牙髓细胞,用荧光定量反转录聚合酶链反应(RT-PCR)检测DSPP、骨钙素mRNA的表达,ALP试剂盒检测ALP活性改变. 结果 诱导分化的牙髓细胞在脂多糖刺激后ALP活性由(1156.10±100.60)pmol·h-1·ng-1下降为(884.80±26.72)pmol·h-1·ng1,荧光定量RT-PCR显示脂多糖能显著抑制DSPP和骨钙素mRNA表达,其中DSPP拷贝数由(176 301.62±13 269.77)下降为(39 396.32±12 018.08),骨钙素拷贝数由(4971.46±483.91)下降为(1104.67±63.37)(t=7.922,P<0.001). 结论 脂多糖能显著降低诱导分化的牙髓细胞合成有机基质.     

aFGF和TGFβ1联合诱导猪牙乳头细胞向成牙本质样细胞分化

目的建立猪牙乳头细胞向成牙本质样细胞分化的体外诱导方案。方法联合应用aFGF和TGFβ1对体外培养的猪牙乳头细胞进行平面诱导,观察诱导后细胞的形态学变化,通过Von Kossa染色检测诱导后细胞的矿化能力,并采用免疫荧光染色和RT-PCR检测成牙本质细胞特异性标志物——DSP蛋白和DSPP mRNA在诱导后细胞中的表达。结果诱导后部分细胞出现细长的单侧细胞突起,在矿化液培养2周后形成典型的矿化结节,并且表达DSP蛋白和DSPP mRNA。结论联合应用aFGF和TGFβ1可以诱导体外培养的猪牙乳头细胞向成牙本质样细胞分化。     

Transforming growth factor-beta1 and basic fibroblast growth factor modulate osteocalcin and osteonectin/SPARC syntheses in vitamin-D-activated pulp cells

Vitamin D deficiency elicits hypocalcified dentin. However, little is known about the action of vitamin D on the syntheses of dentin matrix proteins. In this study, we examined the effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on the expressions of osteocalcin and osteonectin/secreted protein, acidic and rich in cysteine (SPARC), by human pulp cells in the presence or absence of transforming growth factor-beta1 (TGF-beta1) or basic fibroblast growth factor (bFGF). 1,25(OH)2D3 markedly increased osteocalcin at protein and mRNA levels. The osteocalcin level induced by 1,25(OH)2D3 was decreased and increased by TGF-beta1 and bFGF, respectively. 1,25(OH)2D3 suppressed SPARC synthesis at protein and mRNA levels. TGF-beta1, but not bFGF, increased SPARC synthesis in the presence of 1,25(OH)2D3. SPARC, but not osteocalcin, increased DNA synthesis in pulp cells. These findings suggest that 1,25(OH)2D3 and growth factors interactively regulate the expression of osteocalcin and SPARC in pulp cells, and that SPARC can stimulate DNA synthesis by pulp cells.     

过表达骨形态发生蛋白-4对小鼠诱导性多能干细胞生物学活性的影响

目的 构建过表达骨形态发生蛋白-4（BMP4）基因的慢病毒载体,探讨其过表达后对小鼠诱导性多能干细胞（iPS）生物学活性的影响。方法 采用慢病毒转染建立稳定过表达BMP4的iPS细胞株（BMP4过表达组）,未作任何转染的细胞为空白组,转染慢病毒阴性对照组的细胞为空载体组。CCK8检测各组细胞增殖活性,碱性磷酸酶（ALP）活性检测细胞分化程度,荧光定量聚合酶链反应检测BMP4、成釉细胞蛋白（AMBN）、细胞角蛋白（CK）14、牙本质涎磷蛋白（DSPP）、骨唾液酸蛋白（BSP）及骨细胞特异转录因子Runx2的mRNA表达。结果 与空白组及空载体组相比,BMP4过表达组的细胞增殖活性及ALP活性升高（P<0.05）,Runx2、AMBN、CK14、DSPP、BSP mRNA的表达增加（P<0.05）。结论 BMP4基因能够促进iPS的牙向分化。     

Osteoblast proliferation and differentiation on dentin slices are modulated by pretreatment of the surface with tetracycline or osteoclasts

BACKGROUND: Implant surface roughness and chemical composition, as well as other factors, affect the ability of osteogenic cells to form bone adjacent to an implant. The same principles may also apply to the tooth root and some reports have shown that surface modification of the root may lead to improved restoration of the periodontal apparatus. The most common of these surface modification techniques involves demineralization with citric acid or treatment with tetracycline to expose collagen fibrils. In addition, during normal bone remodeling, osteoclasts demineralize the extracellular matrix, leaving resorption pits and exposed collagen fibrils. In this study, the effect of different dentin surface-preparation techniques on osteoblasts were compared. METHODS: Slices of sperm whale dentin were mechanically polished and surfaces were treated with tetracycline-HCl (TCN) or were cultured with mouse bone marrow cells to create a surface with osteoclast (OC) resorption pits or left untreated. Profilometry, x-ray photoelectron spectroscopy (XPS), and scanning electron microscopy (SEM) were used to evaluate the 3 different dentin surfaces. MG63 osteoblast-like cells were cultured on the 3 different surfaces and the effect of dentin surface preparation technique on MG63 cell proliferation (cell number), differentiaton (alkaline phosphatase specific activity of isolated cells and cell layer lysates; osteocalcin production), and local factor production (transforming growth factor (TGF)-beta1 and prostaglandin E2 (PGE2) compared. RESULTS: Profilometry showed the polished and TCN surfaces were smooth with comparable Ra values, whereas the OC surfaces were slightly rougher due to resorption pits which covered 3.7% of the surface. XPS measurements showed that TCN treatment reduced the Ca and P content of the surface, indicating that it had dissolved the mineral. Osteoclast-resorption also reduced the Ca and P content, but to a lesser extent. MG63 cell proliferation on polished dentin and tissue culture polystyrene was equivalent. In contrast, cells grown on the TCN- and OC-treated surfaces exhibited increased proliferation. No effect of surface treatment on cell alkaline phosphatase activity was observed, but activity in the cell layer lysates was increased on the TCN- and OC-treated surfaces. Osteocalcin production was reduced on all dentin surfaces, but the greatest reduction was found on the TCN-treated surface. Production of both TGF-beta1 and PGE2 was increased on the treated surfaces. All effects were greatest in cultures grown on the TCN-treated dentin. CONCLUSIONS: These data indicate that demineralization of the dentin surface promotes proliferation of osteoblasts and early differentiation events like production of alkaline phosphatase and autocrine mediators such as PGE2 and TGF-beta1. However, later differentiation events like osteocalcin production are decreased. Osteoclast-mediated bone resorption elicits similar responses; less than 4% of the dentin surface resulted in approximately 75% of the response caused by TCN treatment. These observations suggest that greater attention should be paid to the effects of osteoclastic resorption in designing methods for enhancing bone and cementum formation adjacent to root surfaces.     

转化生长因子β1对人牙髓干细胞成骨分化作用研究

目的　研究不同浓度的转化生长因子β1（transforming growth factor-β1,TGF-β1）对人牙髓干细胞（human dental pulp stem cells,hDPSCs）成骨分化的影响。方法　采用酶消化法提取hDPSCs,通过流式细胞术鉴定干细胞的表面标记物。实验组利用含有不同浓度TGF-β1（1、5、10、20 ng/mL）的α-MEM培养细胞,对照组以不含TGF-β1的α-MEM培养细胞,分别在第7天和14天时,采用实时荧光定量PCR（qRT-PCR）方法检测成骨相关因子的表达情况。在第21天时,通过茜素红染色观察矿化结节形成情况。结果 　显微镜下可见hDPSCs呈长梭形,形态类似成纤维细胞。流式细胞术检测结果显示细胞呈CD73和CD90阳性表达,CD31、CD34和CD45阴性表达,具有典型的干细胞表面标记物。qRT-PCR结果表明,含有TGF-β1的各实验组均能明显促进成骨相关因子Runt 相关转录因子2（Runx-2）、骨涎蛋白（BSP）和骨钙素（OCN）mRNA的表达。第7天时,TGF-β1浓度为1 ng/mL 组的Runx-2、BSP和OCN的表达水平分别为对照组的11.3倍、8.7倍和11.7倍,明显高于其他各浓度组（P < 0.05）;第14天时,1 ng/mL组的Runx-2、BSP和OCN表达水平分别为对照组的5.08倍、7.17倍和3.03倍（P < 0.05）,20 ng/mL组BSP表达水平与对照组差异无统计学意义（P > 0.05）。茜素红染色显示各浓度的TGF-β1均能促进hDPSCs矿化结节形成。结论　TGF-β1可以促进hDPSCs的成骨分化。     

血管内皮生长因子和转化生长因子β1基因调控人根尖乳头细胞矿化相关因子的研究

目的克隆人血管内皮生长因子（VEGF）异构体中的VEGF165,构建真核表达载体,探讨过表达VEGF165和转化生长因子β1（TGFβ1）对人根尖乳头细胞矿化相关因子的影响。方法提取人脐静脉内皮细胞系ECV304总RNA,采用逆转录聚合酶链反应（RT-PCR）方法扩增VEGF165基因,插入pcDNA3.1hisA,构建重组质粒pcDNA3.1hisA-VEGF165,经酶切及测序验证正确后,将其和pcDNA3.1hisA-TGFβ1转染人根尖乳头细胞,采用实时定量聚合酶链反应检测转染效率和骨涎蛋白（BSP）、牙本质涎磷蛋白（DSPP）、骨钙素（OCN）、牙本质基质蛋白1（DMP1）的表达。结果插入表达载体的VEGF165基因序列与GenBank数据库中的序列具有100%同源性;转染后VEGF165及TGFβ1 mRNA显著增高;各实验组DSPP mRNA的表达均升高（P＜0.05）,实验2组和实验3组的OCN mRNA升高（P＜0.05）,各组间BSP mRNA的表达差异无统计学意义（P＞0.05）,DMP1 mRNA均未见表达。结论成功构建VEGF165真核表达载体,VEGF165和TGFβ1均能促进人根尖乳头细胞多种矿化因子的表达,与根尖乳头细胞的分化相关。     

Transforming growth factor beta2 regulates growth and differentiation of pulp cells via ALK5/Smad2/3

Transforming growth factor beta (TGF-beta) may regulate the biological activities of dental pulp cells. We found that human dental pulp cells expressed TGF-beta1, TGF-beta2, and a little amount of TGF-beta3 messenger RNA (mRNA). The exposure of pulp cells to TGF-beta2 induced the phosphorylation of Smad2/3, Smad1/5/8, and extracellular regulated-kinase 1/2 (ERK1/2) as observed by Western blotting. Exposure to TGF-beta2 decreased the alkaline phosphatase (ALP) mRNA expression and enzyme activity. Pretreatment of pulp cells with SB431542 (an inhibitor of TGF-beta ALK-4, ALK-5, and ALK-7 receptors) but not U0126 (a MEK1 inhibitor) prevented the inhibition of viable cell number, ALP activity, and mRNA expression by TGF-beta2 in dental pulp cells. These results suggest that TGF-beta may affect the growth and differentiation of dental pulp cells via an autocrine fashion by activation of the ALK/Smad2/3-signal transduction pathways. TGF-beta2 possibly regulates the differentiation of pulp cell at specific stages synergistically with other factors.     

Platelet-rich plasma contains high levels of platelet-derived growth factor and transforming growth factor-beta and modulates the proliferation of periodontally related cells in vitro

BACKGROUND: Platelet-rich plasma (PRP) is a fraction of plasma, in which platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta) are thought to be concentrated. It is plausible that topically-applied PRP up-regulates cellular activity and subsequently promotes periodontal regeneration in vivo. However, the concentrations of these growth factors in PRP have not been specifically determined and the biological effects of PRP at the cellular and molecular levels have not been determined. METHODS: PRP obtained from 20 healthy subjects was prepared from plasma by centrifugation. These PRP preparations were immediately subjected to an evaluation for PDGF-AB and TGF-beta1 using enzyme-linked immunosorbent assay (ELISA) kits. The biological effects of the PRP preparations were evaluated on osteoblastic, epithelial, fibroblastic, and periodontal ligament cells. Cellular mitogenic activity was evaluated by counting cell numbers or evaluating 5-bromodeoxyuridine (BrdU) incorporation. Expression of alkaline phosphatase (ALP) was immunocytochemically evaluated. RESULTS: In the PRP preparations, platelets were concentrated up to 70.9 x 10(4) cells/microl (283.4% of the unconcentrated plasma). The levels of PDGF-AB and TGF-beta1 were also concentrated up to 182.0 ng/ml (440.6%) and 140.9 ng/ml (346.6%), respectively. Scatter plots revealed significant correlations between platelet counts and levels of these growth factors. PRP stimulated osteoblastic DNA synthesis and cell division (138% of control), with simultaneous down-regulation of ALP, but suppressed epithelial cell division (80% of control). PRP also stimulated DNA synthesis in gingival fibroblasts and periodontal ligament cells. CONCLUSIONS: These data demonstrated that both PDGF-AB and TGF-beta1 were highly concentrated in the PRP preparations. It is suggested PRP modulates cell proliferation in a cell type-specific manner similar to what has been observed with TGF-beta1. Since synchronized behavior of related cell types is thought to be required for successful periodontal regeneration, it is further suggested these cell type-specific actions may be beneficial for periodontal regenerative therapy.     

可注射型富血小板纤维蛋白对人根尖牙乳头干细胞生物学行为的影响

目的 探讨可注射型富血小板纤维蛋白（injectable platelet rich fibrin，iPRF）对人根尖牙乳头干细胞（human stem cells from apical papilla，hSCAPs）生物学行为的影响。方法 酶联免疫吸附实验（enzyme linked immunosorbent assay，ELISA）测定可注射型富血小板纤维蛋白提取液（injectable platelet rich fibrin extract，iPRFe）生长因子的含量；CCK 8、Transwell实验、茜素红染色分别检测iPRFe对hSCAPs增殖、迁移、矿化诱导的影响；荧光实时定量PCR检测与成骨/成牙向分化相关基因的mRNA表达水平。结果 iPRFe中可检测到血小板衍生生长因子 BB（platelet derived growth factor BB，PDGF BB)、胰岛素样生长因子1（insulin like growth factor 1，IGF1）和转化生长因子β1（transforming growth factor β1，TGFβ1）；iPRFe能促进hSCAPs的增殖，且在1/4×浓度时hSCAPs增殖效果最佳；1/4×浓度的iPRFe对hSCAPs的迁移和矿化也有明显的促进效果；iPRFe还可明显促进碱性磷酸酶（alkaline phosphatase，ALP）、牙本质基质蛋白1（dentin matrix protein 1，DMP1）、牙本质涎磷蛋白（dentin sialophosphoprotein，DSPP）和骨钙素（osteocalcin，OCN） mRNA的表达（P<0.05）。结论 iPRF能够促进hSCAPs的增殖、迁移和矿化，有望为牙髓再生提供可能。     

Hypoxia promotes mineralization of human dental pulp cells

Introduction

Dental pulp can be exposed to hypoxic conditions in case of trauma or inflammation. Dental pulp cells (DPCs) have mineralization potential, which plays a key role in pulp repair and reparative dentinogenesis process. Little information is available about DPC mineralization in hypoxic condition. The purpose of this study was to assess the influence of hypoxia on DPC mineralization to pave the way for a better understanding of dental pulp regeneration and reparative dentin formation.

Methods

Human DPCs were obtained by using tissue explant technique in vitro and cultured in normoxia (20% O₂) or hypoxia (5% O₂). Cell viability was investigated by methyl-thiazol-tetrazolium assay. Cell mineralization was assessed by von Kossa staining and alizarin red S staining. Important mineral genes such as osteocalcin (OCN), dentin matrix acidic phosphoprotein-1 (DMP-1), bone sialoprotein (BSP), and dentin sialophosphoprotein (DSPP) were determined by real-time polymerase chain reaction.

Results

Cell viability of DPCs increased more in hypoxia than in normoxia from day 3 to day 5. Von Kossa staining and alizarin red S staining showed DPCs in hypoxia had higher mineralization activity than in normoxia. Expression of mRNAs for OCN, DMP-1, BSP, and DSPP was greater in hypoxia than in normoxia.

Conclusions

These results imply that hypoxia promotes DPC mineralization.