二甲胺四环素促进骨髓基质细胞增殖作用的研究

为弄清二甲胺四环素增加摘除卵巢的老龄大鼠骨量作用的细胞分子基础,采用细胞增殖曲线的绘制和碱性磷酸酶测定方法,了解二甲胺四环素对成骨细胞和骨髓基质细胞的影响,结果：二甲胺四环素在体外刺激骨髓基质细胞和成骨细胞增殖的作用,但抑制细胞的分化。     

白细胞介素10对体外培养大鼠主动脉平滑肌细胞成骨样分化 与钙化的影响

目的 探究白细胞介素10(IL-10)对体外培养大鼠主动脉平滑肌细胞(VSMCs)成骨分化,钙化和可能的信号传导途径。方法 提取大鼠胸VSMCs,采用钙浓度测定,碱性磷酸酶(ALP)活性测定和茜素红染色观察IL-10对钙化的影响。实时聚合酶链反应(Real-Time PCR)探究IL-10对成骨样分化的作用;蛋白质免疫印迹(Western Blot)用于检测成骨分化蛋白,观察IL-10对高钙高磷诱导的VSMCs钙化的作用。结果 IL-10促进高钙高磷引起的VSMCs钙化,上调成骨分化标志物的表达,激活骨形态形成蛋白2/白细胞抑制因子1,5/Runt相关转录因子2通路(BMP2/Smad1,5/RUNX2),并且抑制活化T细胞核因子c1(NFATc1)的表达。结论 IL-10能够诱导VSMCs成骨样分化,这可能是临床上观察到IL-10与血管钙化相关的机制之一。IL-10这一作用与其激活BMP2/Smad1,5/RUNX2通路,抑制NFATc1激活有关。     

毛蕊花糖苷对新生大鼠体外培养成骨细胞增殖与分化作用研究

目的研究毛蕊花糖苷对体外培养新生大鼠颅骨成骨细胞(rat calvarial osteoblasts,ROB)增殖、分化作用的影响。方法采用Ⅱ型胶原酶消化法分离制备新生大鼠颅骨ROB细胞,MTT法测定ROB细胞增殖,碱性磷酸酶(ALP)试剂盒法测定ROB细胞内ALP活性,以细胞的增殖率和ALP活性作为考察指标,观察不同浓度的毛蕊花糖苷对ROB细胞增殖和分化作用的影响。结果 1×10-7～1×10-9mol·L-1的毛蕊花糖苷对ROB细胞的增殖具有显著的促进作用(P<0.05),且终浓度为1×10-7mol·L-1的毛蕊花糖苷在作用72h后能显著提高ROB细胞内碱性磷酸酶的活性(P<0.05)。结论一定浓度的毛蕊花糖苷能显著的促进ROB细胞的增殖和分化。     

淫羊藿苷对体外在无地塞米松培养基中诱导培养大鼠骨髓基质细胞成骨性分化的影响

目的：探讨在无地塞米松（dexamethasone）时淫羊藿苷（icariin,ICA）对体外培养大鼠骨髓基质细胞成骨性分化的影响,并试图寻找一种新的或更强的促体外培养骨髓基质细胞成骨性分化的诱导剂。方法用1×10－5 mol·L －1淫羊藿苷或1×10－8 mol·L －1的地塞米松对大鼠骨髓基质细胞分别进行药物干预,细胞计数试剂盒（Cell Counting Kit-8,CCK-8）检测细胞增殖,碱性磷酸酶检测试剂盒检测碱性磷酸酶（alkaline phosphatase,ALP）活性,茜素红染色检测钙化结节数量,实时定量聚合酶链反应（RT-PCR）检测骨保护素（osteoprotegerin,OPG）、核因子-κB 受体活化因子配体（receptor activator of NF-κB ligand, RANKL）、骨形态发生蛋白（bone morphogenetic protein,BMP）的信使 RNA（mRNA）表达情况。结果1×10－5 mol·L －1淫羊藿苷在无地塞米松的培养基中能显著提高大鼠骨髓基质细胞 ALP 活性;明显增加大鼠骨髓基质细胞钙化结节数目;显著上调BMP、OPG mRNA 水平以及 OPG／RANKL mRNA 比值。结论淫羊藿苷可能是一种潜在的促体外培养大鼠骨髓基质细胞成骨性分化的新诱导剂。     

寡核苷酸促人牙周膜细胞增殖及成骨分化的实验研究

目的研究寡核苷酸(ODN)mt01对人牙周膜细胞增殖和向成骨细胞分化的影响,为临床药剂的开发奠定实验基础。方法采用人离体牙牙周膜细胞作为研究对象,原代培养牙周膜细胞,确定细胞最佳铺板浓度后,通过四甲基偶氮唑盐(MTT)比色法研究ODN mt01对人牙周膜细胞增殖的影响;用碱性磷酸酶(ALP)试剂盒检测ODN mt01影响人牙周膜细胞成骨分化的最佳工作浓度。结果分别作用于人牙周膜细胞24、48、72和96 h,ODN mt01在整个作用过程中均表现出显著的促人牙周膜细胞增殖作用(P<0.05);与磷酸盐缓冲液(PBS)对照组ALP表达量相比,工作浓度为1.0 mg/L时,ODN mt01可促进人牙周膜细胞表达ALP水平显著升高(P<0.05)。结论适当工作浓度的ODN mt01可以促进体外培养的人牙周膜细胞增殖和成骨分化。     

磷酸化cAMP反应元件结合蛋白对发育期人牙乳头细胞增殖和分化的影响

目的 构建CBP抑制表达慢病毒载体,探讨CBP有效表达沉默后对体外培养人牙乳头细胞(human dental papilla cells,HDPC)的增殖及分化影响.方法 利用基因重组技术构建重组慢病毒建立牙乳头细胞沉默稳转细胞,采用CCK-8检测细胞增殖的变化,采用检测分化相关蛋白的表达评价CBP对HDPC分化能力的影响.结果 测序结果提示成功构建四组人CBP基因的重组慢病毒;转染HDPC后,通过RT-PCR和蛋白质印迹法筛选获得有效靶点的重组慢病毒;CCK-8检测结果表明HDPC在CBP表达下调后增殖活性受到抑制;慢病毒介导CBP沉默后影响了HDPCs相关分化蛋白ColI、OCN、OPN的表达,较对照组显著下调(P＜0.05);牙乳头细胞的ALP活性在转染重组慢病毒后5,7,14 d后显著降低(P＜0.05);结论 成功构建了高效CBP抑制表达载体,CBP沉默可抑制HDPCs的增殖和分化.     

马氏珠母贝糖胺聚糖对体外成骨细胞增殖、分化及矿化功能的影响

目的观察珠母贝糖胺聚糖在体外对新生大鼠颅骨成骨细胞增殖、分化及矿化功能的影响。方法应用MTT法、PNPP法、茜素红染色 (ARS)矿化骨结节及用图像分析仪计算骨结节的面积等方法观察细胞的增殖、碱性磷酸酶 (ALP)活性及矿化结节形成的影响。结果珠母贝糖胺聚糖 0 .0 0 8～ 0 .5 g·L- 1在不同时间MTT测得的A值均低于空白对照组 ,不促进细胞的增殖 ;各浓度组在培养 5d时 ,碱性磷酸酶的活性显著高于空白对照组 ;培养 31d后茜素红染色显示 ,0 .0 6 3g·L- 1组所形成的骨结节面积显著大于空白对照组。结论珠母贝糖胺聚糖对体外培养的SD新生大鼠颅骨成骨细胞有显著促进分化和矿化的作用 ,但不促进细胞的增殖。     

SHH对体外培养的小鼠牙乳头间充质细胞增殖的影响

目的 观察Sonic Hedgehog对体外培养小鼠牙乳头间充质细胞增殖的影响。方法 构建Shh真核表达载体pCDNA3-Shh，脂质体法转染COS7细胞，收集培养上清波。实验组加入转染pCDNA3-Shh的培养上清液，对照组加入转染空栽体pCDNA3的培养上清液，培养72h，MTT法检测Shh对体外培养的小鼠牙乳头间充质细胞增殖的影响。结果 Shh能促进体外培养的小鼠牙乳头间充质细胞增殖。结论 Shh促进牙孔头细胞增殖，参与牙胚发育。     

补肾方剂对大鼠骨髓基质干细胞增殖与分化的影响

目的观察补肾方剂对体外培养SD大鼠骨髓基质干细胞细胞增殖、分化及矿化的影响。方法应用四甲基偶氮唑盐法、硝基苯磷酸盐法及茜素红染色方法观察不同浓度补肾方剂对体外培养骨髓基质干细胞在1、2、3、4、5d时的增殖、碱性磷酸酶(ALP)活性及矿化结节形成的影响。结果不同浓度的补肾方均可促进骨髓基质干细胞增殖率提高,以中、高浓度组的作用较为明显(P<0.01);不同浓度补肾方剂可使ALP活性增强(P<0.01);中、高浓度组补肾活血方矿化结节数量显著多于对照组(P<0.01)。结论补肾方剂具有促进骨髓基质干细胞的增殖、分化及矿化的作用。     

阿伦磷酸钠对小鼠骨髓基质细胞成骨和成脂分化的影响

目的在离体条件下研究阿伦磷酸钠对小鼠骨髓基质细胞(BMSCs)成骨和成脂分化的影响。方法采用四甲基偶氮唑盐(MTT)法检测阿伦磷酸钠对骨髓基质细胞增殖的影响,碱性磷酸酶(ALP)活性测定法检测阿伦磷酸钠对骨髓基质细胞(MSCs)成骨分化的影响,油红O染色形态学及定量测定法检测阿伦磷酸钠对骨髓基质细胞成脂分化的影响。结果1×10-10,1×10-9,1×10-8,1×10-7,1×10-6和1×10-5mol.L-1的阿伦磷酸钠可以促进小鼠原代骨髓基质细胞增殖,抑制骨髓基质细胞的成骨分化及成脂分化。结论阿伦磷酸钠对骨质疏松症的预防和治疗作用可能通过抑制骨髓基质细胞的成脂分化,进而减少脂肪细胞细胞因子的分泌而抑制破骨细胞的形成、激活以及骨吸收功能。     

1,25(OH)2D3在牙乳头干细胞成骨分化期的作用研究

摘要 目的：研究1,25(OH)2D3在牙乳头干细胞向成骨细胞分化过程中的作用角色。方法：分离培养牙乳头干细胞,培养基中添加不同浓度1,25(OH)2D3,MTT法检测细胞生长速度,流式细胞术检测细胞增殖周期变化,western blot方法检测核因子-k B受体活化剂配体（RANKL）、骨保护素（OPG）和维生素D受体（VDR）的蛋白质表达水平;siRNA技术沉默VDR表达后,检测其对RANKL和OPG蛋白质表达的影响。结果：MTT和流式细胞术检测结果显示1,25(OH)2D3对牙乳头干细胞的增殖没有明显作用;western blot结果显示RANKL、OPG和VDR的蛋白质表达与1,25(OH)2D3浓度具有剂量相关性;siRNA沉默VDR表达后,RANKL和OPG的蛋白质表达水平均有不同程度下降。结论：1,25(OH)2D3通过调节VDR水平影响牙乳头干细胞向成骨细胞方向的分化过程。     

Promotion effect of acankoreoside J, a lupane-triterpene in Acanthopanax koreanum, on hair growth

This study was conducted to evaluate the effect of Acanthopanax koreanum and acankoreoside J from A. koreanum on the promotion of hair growth. When immortalized rat vibrissa dermal papilla cells were treated with extract of A. koreanum leaves, the proliferation of dermal papilla cells significantly increased. In particular, acankoreoside J among several components, isolated from A. koreanum leaves, markedly promoted the proliferation of the dermal papilla cells. When rat vibrissa follicles were treated with an acankoreoside J, the hair-fiber lengths of the vibrissa follicles increased significantly. We further investigated β-catenin pathway and cell cycle regulation with respect to the effect of acankoreoside J on the proliferation of the dermal papilla cells. Treatment with acankoreoside J results in an increase of nuclear β-catenin level, and up-regulation of cyclin D1, cyclin E and CDK2, whereas, the expression of p27(kip1) was down-regulated in the dermal papilla cells. Taken together, these results suggest that acankoreoside J, a lupane-triterpene of A. koreanum, has the potential of promoting hair growth by promoting cell cycle progression of the dermal papilla cells, through the increase of nuclear β-catenin, along with the up-regulation of cyclin D1, cyclin E and CDK2, and down-regulation of p27(kip1).     

Changes in matrix extracellular phosphoglycoprotein expression before and during in vitro osteogenic differentiation of human dental papilla mesenchymal cells

The purpose of this study is to characterise the expression of matrix extracellular phosphoglycoprotein (MEPE) in cultured mesenchymal cells isolated from human dental papilla (PaMCs) of impacted third molars either before or during differentiation of these cells into osteo/odontoblasts. PaMCs, like mesenchymal cells deriving from human dental pulp (DPMCs), resulted positive for a number of mesenchymal markers including CD146 and STRO-1. During the first week in culture they showed a faster proliferation rate than DPMCs, coupled to an earlier down-regulation of MEPE. Also when the cells were further cultured in osteogenic medium (containing beta-glycerophosphate, ascorbic acid and dexamethasone) for 40 days, MEPE down-regulation coupled to an increased expression of osteogenic markers, such as osteocalcin and alkaline phosphatase, occurred earlier in PaMCs than in DPMCs. Thus, our data, indicating that also in PaMCs MEPE expression is higher when cells proliferate, whereas it is downregulated as cells differentiated, are in favour of a role of MEPE as an early regulator of odontogenic differentiation. We also confirm the superior proliferative potential of PaMCs in comparison with DPMCs, coupled to a more rapid induction of osteogenic differentiation. Therefore, these cells represent an optimal source to be conveniently used for dental tissue engineering and tooth regeneration.     

Salidroside stimulates osteoblast differentiation through BMP signaling pathway

Salidroside (SAL) is one of main active components of Rhodiola rosea L. and possesses diverse pharmacological effects. However, the direct role of SAL in bone metabolism remains elusive. In this study, effects of SAL on osteoblast differentiation of murine pluripotent mesenchymal cell line C3H10T1/2 and osteoblastic cell line MC3T3-E1 were examined. We first identified SAL as a potential BMP2 activator in a cell-based screening assay. SAL (0.5–10 μM) could slightly promote the proliferation and greatly increase the alkaline phosphatase (ALP) activity in both cells. Furthermore, SAL increased the mRNA expressions of osteoblast marker genes in either C3H10T1/2 or MC3T3-E1 cells after treatment for different time. Moreover, the mineralization of C3H10T1/2 cells assayed by Alizarin red S staining was dose-dependently increased by SAL. Mechanistically, SAL increased the mRNA level of genes involved in the regulation of BMP signaling pathway, including BMP2, BMP6 and BMP7 and enhanced the phosphorylation of Smad1/5/8 and ERK1/2. The osteogenic effect of SAL was abolished by BMP antagonist noggin or by BMP receptor kinase inhibitor dorsomorphin. Further in vivo study demonstrated that SAL reversed bone loss in ovariectomized rats. Collectively, our findings indicate that SAL regulates bone metabolism through BMP signaling pathway.     

Bone morphogenetic protein signalling is required for the anti-mitogenic effect of the proteasome inhibitor MG-132 on colon cancer cells

BACKGROUND AND PURPOSE: Inhibition of proteasome has been emerging as a promising approach in pathway-directed cancer therapy. Bone morphogenetic protein (BMP) signalling, which is known to be regulated by the ubiquitin-proteasome pathway in osteoblasts, plays a crucial role in the suppression of gastrointestinal carcinogenesis. Here we sought to elucidate the anti-mitogenic effect of a proteasome inhibitor in relation to BMP signalling in colon cancer. EXPERIMENTAL APPROACH: The effects of the proteasome inhibitor MG-132 on proliferation of SW1116 and HT-29 colon cancer cells were determined by [(3)H]-thymidine incorporation and colony-formation assay. The involvement of BMP signalling in the action of MG-132 was elucidated by western blot, real-time PCR, immunofluorescence and RNA interference. KEY RESULTS: MG-132 significantly suppressed the proliferation of colon cancer SW1116 and HT-29 cells. In this regard, MG-132 activated BMP signalling and this was manifested as an increase in Smad1/5/8 phosphorylation and upregulation of p21(Waf1/Cip1) and p27(Kip1) expression. Knockdown of BMP receptor II abolished Smad1/5/8 phosphorylation, the induction of p21(Waf1/Cip1) and p27(Kip1) and inhibition of cell proliferation induced by MG-132. Further analysis revealed that MG-132 upregulated the expression of BMP1 and BMP2, which are secreted members of the BMP superfamily. Moreover, the expression of Smad6, an intracellular inhibitor of BMP signalling, was suppressed by MG-132. CONCLUSIONS AND IMPLICATIONS: These findings suggest that inhibition of proteasome suppresses the proliferation of colon cancer cells via activation of BMP signalling. They also demonstrate a novel aspect of proteasome function in the regulation of colon cancer cell proliferation.     

Gene expression profile of dental pulp cells during differentiation into an adipocyte lineage

Gene regulation during in vitro differentiation into adipocytes was examined in rat dental pulp-derived cells. Insulin, 3-isobutyl-1-methylxanthine, and dexamethasone were added to induce adipogenesis. Cells containing lipid droplets were observed after induction as in 3T3 L1 cells. Rat dental pulp-derived cells showed their potential to differentiate into adipocytes in vitro. In both types of cells, the pluripotent markers Oct-3/4 and Sox2 were downregulated during differentiation, whereas the expression of Nanog was not significantly changed during differentiation. Interestingly, in the dental pulp-derived cells, the level of Oct-3/4 was transiently induced at 1 week after induction and then significantly decreased during differentiation. Based on the expression profiles determined using GeneChip Arrays, 3418 probes across 10 clusters showed a difference in expression at 1, 2, and 3 weeks after induction versus before induction. Notably, genes in the PPAR signaling pathway including Pparγ, Fabp4, and the C/EBP family were upregulated by more than 3-fold. Upregulation of the PPAR pathways seems to be a critical signal transduction pathway in this differentiation system. These findings indicate that dental pulp-derived cells are a potential source of adipogenic cells, and their gene expression profile could be useful in future regenerative medicine applications.     

Nicotinamide phosphoribosyltransferase regulates the cell differentiation and mineralization in cultured odontoblasts

The aim of the present study was to investigate the physiological role of nicotinamide phosphoribosyltransferase (NAMPT) associated with odontogenic differentiation during tooth development in mice. Mouse dental papilla cell-23 (MDPC-23) cells cultured in differentiation media were stimulated with the specific NAMPT inhibitor, FK866, and Visfatin (NAMPT) for up to 10 days. The cells were evaluated after 0, 4, 7, and 10 days. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The mineralization assay was performed by staining MDPC-23 cells with Alizarin Red S solution. After cultivation, MDPC-23 cells were harvested for quantitative PCR or Western blotting. Analysis of variance was performed using StatView 5.0 software (SAS Institute Inc., Cary, NC, USA). Statistical significance was set at p < 0.05. The expression of NAMPT increased during the differentiation of murine odontoblast-like MDPC-23 cells. Furthermore, the up-regulation of NAMPT promoted odontogenic differentiation and accelerated mineralization through an increase in representative odontoblastic biomarkers, such as dentin sialophosphoprotein, dentin matrix protein-1, and alkaline phosphatase in MDPC-23 cells. However, treatment of the cells with the NAMPT inhibitor, FK866, attenuated odontogenic differentiation, as evidenced by the suppression of odontoblastic biomarkers. These data indicate that NAMPT regulated odontoblastic differentiation through the regulation of odontoblastic biomarkers. The increase in NAMPT expression in odontoblasts was closely related to the formation of the extracellular matrix and dentin via the Runx signaling pathway. Therefore, these data suggest that NAMPT is a critical regulator of odontoblast differentiation during tooth development.     

Characterization and biodistribution of human mesenchymal stem cells transduced with lentiviral-mediated BMP2

Recently, the genetic modification of mesenchymal stem cells (MSCs) has led to increased differentiation potential. For the therapeutic application of genetically modified MSCs, it is crucial to evaluate their characteristics and safety. In this study, we investigated the effects of bone morphogenetic protein 2 (BMP2) gene transfer on the characteristics and biodistribution of human MSCs. Lentiviral-mediated BMP2 transduction to MSCs enhanced osteocyte differentiation and decreased adipocyte differentiation. Although there is no significant difference in cell proliferation capacity, MSCs transduced BMP2 proliferate somewhat higher than nontransduced or GFP transduced MSCs. No significant changes were observed in surface antigen expression in genetically modified MSCs. In vivo transplantation of lentiviral-mediated BMP2 gene transferred MSCs to nude mice did not result in tumor formation. To evaluate the biodistribution of genetically modified cells, MSCs carrying BMP2 were injected into the tail vein of femur fractured mice. The introduced MSCs were detected in the spleen, testis and fractured femur 28 days post-implantation. These findings suggest that diverse safety tests for genetically modified MSCs should be considered, particularly when a lentivirus mediated gene transfer method is used.