不同浓度淫羊藿苷修复骨损伤:争议与探索

背景:作为淫羊藿主要有效成分之一的淫羊藿苷,对于骨损伤后的修复具有重要作用。淫羊藿苷在体内的持续有效浓度对于骨损伤的修复至关重要。目的:综述近年国内外淫羊藿苷对于骨修复的研究进展,探讨淫羊藿苷的药理活性浓度。方法:第一作者应用计算机检索2000年1月至2013年10月PubMed数据(http://www.ncbi.nlm.nih.gov/PubMed)及CNKI中国期刊全文数据库(http://www.cnki.net/)有关淫羊藿苷对骨组织修复作用,或者与淫羊藿苷治疗骨损伤的相关基础研究文献。英文检索词"icariin,concentration,bone";中文检索词"淫羊藿苷,浓度,骨"。排除重复性研究,共检索到76篇相关文献,44篇文献符合纳入标准。结果与结论:利用淫羊藿苷的有效活性成分联合缓释支架材料,可以诱导骨髓间充质干细胞成骨分化,增强成骨细胞活性,抑制破骨细胞的吸收,从而起到修复骨组织的作用。淫羊藿苷促进细胞分化作用虽已得到肯定,但是否具有促进细胞增殖作用还存在一定的争议。研究发现,淫羊藿苷的药理活性浓度从10-8到10-5 mol/L不等,总体来看,10-8-10-5 mol/L应该是淫羊藿苷发挥其最佳药理活性可能出现的区间,但目前临床试验尚未开展,具体的持续给药浓度还不确定,这些都需要进一步研究探索。     

淫羊藿苷促进骨折愈合作用机制的研究进展

近年来 , 国内外学者对中药淫羊藿主要有效成分淫羊藿苷加速骨折愈合的机制做了一系列研究,目前研究结果显示, 淫羊藿苷加速骨折愈合的机制主要是通过激活 Wnts、BMP、MAPK、ER 等相关信号通路促进 BMSCs、ASCs 的迁移归巢,进行成骨性分化为成骨细胞,同时促进成骨细胞的成熟、矿化以形成骨痂,并抑制破骨细胞的骨吸收活性。本文从淫羊藿苷对相关细胞以及对血管重建的作用方面对淫羊藿苷加速骨折愈合的作用机制做一回顾综述,以期为后续的研究提供参考。     

补肾中药成分配伍对成骨细胞成骨活性及Smad4 mRNA表达的影响

背景：补肾中药对动物及细胞实验研究提示具有防治骨质疏松及改善骨代谢作用,但补肾中药的配伍研究较少且复杂,且有效作用成分之间的相互作用及药物物质基础尚不确定,筛选最佳配伍困难。 目的：通过采用不同补肾中药成分配伍,对新生SD大鼠成骨细胞进行干预性培养,并对其增殖、分化、Smad4 mRNA表达进行测定,从而筛选并确定补肾中药的最佳配伍。 方法：第5代成骨细胞分为5组：淫羊藿苷组细胞中加入1×10^-5 mol/L的淫羊藿苷;淫羊藿苷+柚皮苷组细胞中加入1×10^-5 mol/L淫羊藿苷+1×10^-5 mol/L柚皮苷;淫羊藿苷+薯蓣皂苷元组细胞中加入1×10^-5 mol/L淫羊藿苷+1×10^-5 mol/L薯蓣皂苷元;淫羊藿苷+梓醇组细胞中加入1×10^-5 mol/L淫羊藿苷+1×10^-5 mol/L梓醇;对照组细胞中加入10 μL生理盐水。每组6个复孔。 结果与结论：与对照组相比,淫羊藿苷+柚皮苷组和淫羊藿苷+薯蓣皂苷元组成骨细胞增殖能力及Smad4 mRNA表达水平增加,且培养72 h时,淫羊藿苷+柚皮苷组成骨细胞增殖能力最强。48 h时,淫羊藿苷+柚皮苷组和淫羊藿苷+薯蓣皂苷元组成骨细胞碱性磷酸酶活性高于对照组;72 h,淫羊藿苷+柚皮苷组和淫羊藿苷+梓醇组成骨细胞碱性磷酸酶活性高于对照组。提示淫羊藿苷等补肾中药成分配伍可以影响成骨细胞的成骨活性,且其中以淫羊藿苷配伍柚皮苷的作用最强。 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     

淫羊藿对骨髓间充质干细胞成骨分化的影响

背景:中药淫羊藿一直以来被用于骨质疏松的治疗和骨缺损的修复。目的:探讨淫羊藿对骨髓间充质干细胞成骨性分化的影响。方法:应用数据库文献检索的方法获取淫羊藿对骨髓间充质干细胞成骨性分化影响研究的文献,对符合研究标准的文献进行深入的分析。通过检测淫羊藿诱导骨髓间充质干细胞成骨性分化过程中碱性磷酸酶、骨钙素、骨桥蛋白、转化生长因子β、骨形态发生蛋白、骨钙素、骨涎蛋白等,了解淫羊藿对骨髓间充质干细胞的增殖和成骨分化的机制。结果与结论:淫羊藿对骨髓间充质干细胞的影响呈剂量依赖性,在诱导培养的早期淫羊藿苷可以提高细胞的磷酸酶活性,在诱导培养的晚期增加细胞钙化结节数,促进骨钙素分泌量,显著提高转化生长因子β1、骨形成蛋白2、胰岛样生长因子1、骨桥蛋白和骨涎蛋白等的表达水平。淫羊藿对骨髓间充质干细胞的增殖和成骨性分化有促进作用,可作为一种良好的骨诱导活性因子。     

淫羊藿对骨的作用研究进展

淫羊藿 (HerbaEpimdii)为小檗科植物 ,又名仙灵脾、三枝九叶草等 ,具有“补肾壮阳 ,益精健骨”之功效。淫羊藿多糖、淫羊藿苷、淫羊藿甙和黄酮类为其主要成份[1 ] 。淫羊藿的作用广泛 ,如对心血管系统、中枢神经系统、血液系统、免疫系统、抗炎、抗骨质疏松、抗衰老、抗肿瘤等均有作用 ,本文就其对骨的作用的研究现状作一综述。一、淫羊藿对成骨细胞增殖与分化的影响成骨细胞是蛋白分泌型细胞 ,能产生Ⅰ型胶原 ,合成分泌骨基质 ,具有高碱性磷酸酶 (ALP)活性 ,并能吸收和转运钙离子 ,是骨形成和骨再建的重要功能细胞。目前认为 ,ALP活性…     

可注射温敏淫羊藿苷壳聚糖水凝胶促进家兔桡骨骨折的愈合

背景：淫羊藿可显著促进体外培养成骨细胞的增殖、分化和成熟,刺激骨形成。 目的：观察可注射温敏淫羊藿苷壳聚糖水凝胶修复家兔桡骨骨折愈合的效果。 方法：麻醉下将36只兔左右桡骨截断,随机分为实验组、对照组和空白组,前两组分别在骨折端周围注入可注射温敏携淫羊藿苷壳聚糖水凝胶、壳聚糖水凝胶,空白组骨折区未注入任何材料。 结果与结论：3组骨折均于术后6周时愈合。X射线片显示,术后2,4, 6周,实验组骨折修复区单位面积平均灰度值及成骨细胞计数均明显高于对照组、空白组(P < 0.05),且实验组成骨细胞增生活跃。说明可注射温敏携淫羊藿苷壳聚糖水凝胶具有促成骨活性,可促进骨折愈合。     

淫羊藿苷治疗骨不连的分子机制

文题释义： 骨不连：骨组织具有强大的自我修复能力，给予适当治疗后大多数骨折都会很快很好地愈合。然而，仍有一小部分骨折难以愈合，当骨折超过3个月仍未愈合，称为延迟愈合，当骨折超过9个月仍未达到骨性连接，则称为骨不连。 淫羊藿苷：为淫羊藿干燥茎叶的提取物，呈淡黄色针状结晶粉末，相对分子质量为676.65，属黄酮类化合物，现代药理学研究发现其具有很强的生物活性，对骨组织、免疫系统、肿瘤组织、神经系统、生殖系统、内分泌系统和心血管系统等具有显著作用。 背景：骨不连是骨科临床上常见的并发症，严重影响患者的身心健康和生活质量。近年来大量的研究发现淫羊藿苷在促进骨折愈合和治疗骨缺损方面有着显著作用；骨不连和骨折愈合是对立共存的，有关骨折愈合机制的研究实际上就是治疗骨不连的研究。 目的：综述淫羊藿苷在骨不连治疗过程中的分子机制研究进展。 方法：第一作者应用计算机以“icariin，bone nonunion，bone marrow mesenchymal stem cells，periosteal cell，osteoblasts，osteoclast”及“淫羊藿苷、骨不连、骨髓间充质干细胞、骨膜细胞、成骨细胞、破骨细胞”作为主题词检索PubMed、中国知网、万方、维普数据库，共检索到542篇文献，按入选标准及排除标准进行筛选，最终纳入44篇文献。结果与结论：淫羊藿苷通过促进骨髓间充质干细胞和骨膜细胞增殖及向成骨细胞分化、促进成骨细胞增殖与成熟、抑制破骨细胞的破骨作用，有效促进骨折愈合和治疗骨不连。目前绝大部分研究仍处于基础实验阶段，尚需要大量临床研究，对于相关蛋白及基因方面也有待于进一步研究，希望为骨不连的中药治疗提供新思路。 ORCID: 0000-0002-2013-743X(余绍涌) 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     

淫羊藿苷对大鼠间充质干细胞骨向分化过程中转化生长因子β_1、骨形态发生蛋白2表达的影响

背景:淫羊藿苷作为补肾中药淫羊藿的主要有效成分,能够有效促进间充质干细胞向成骨细胞分化。目的:观察淫羊藿苷在诱导间充质干细胞骨向分化过程中对转化生长因子β1、骨形态发生蛋白2表达的影响,阐释其可能的诱导机制。方法:运用全骨髓贴壁法分离、纯化大鼠骨髓间充质干细胞;以20mg/L淫羊藿苷作为间充质干细胞药物干预浓度,根据干预条件的不同将实验分为:空白组、经典组(加经典成骨诱导剂)、淫羊藿苷组、淫羊藿苷+经组;ELISA法检测各组转化生长因子β1、骨形态发生蛋白2的表达。结果与结论:经典组、淫羊藿苷组、淫羊藿苷+经组转化生长因子β1、骨形态发生蛋白2值均高于空白组(P0.05);各组第7天的转化生长因子β1值高于14,21d(P0.01),各组不同诱导时间骨形态发生蛋白2值之间相比差异无显著性意义(P0.05)。提示上调转化生长因子β1、骨形态发生蛋白2的表达量可能是淫羊藿苷诱导各组促进间充质干细胞骨向分化的作用机制之一。     

淫羊藿苷调节酸性微环境减轻绝经后老年骨质疏松性疼痛

背景：前期研究已证明，淫羊藿苷在促进骨形成和抑制骨吸收方面具有重要作用，但其对骨质疏松介导的骨痛产生的影响尚未见报道。目的：探讨淫羊藿苷减轻绝经后老年性骨质疏松性骨痛的可能机制。方法：(1)动物实验：将200只C57BL/6小鼠随机分为4组：假手术组、模型组、模型+淫羊藿苷组，模型+碳酸酐酶Ⅱ抑制剂组。除假手术组外，其余各组摘除小鼠卵巢建立绝经后骨质疏松模型。模型+淫羊藿苷组在造模后第2天灌胃淫羊藿苷，每2周进行1次疼痛行为学实验并取材，持续20周。microCT检测股骨骨量、苏木精-伊红染色及TRAP染色检测破骨细胞活性、免疫荧光染色检测神经元形态及相关离子通道表达。(2)细胞实验：提取小鼠骨髓来源的破骨细胞前体细胞，在体外使用RANKL/M-CSF体系诱导分化为破骨细胞并添加不同浓度淫羊藿苷(1,10μmol/L)干预。采用抗酒石酸酸性磷酸酶染色检测破骨细胞分化，采用鬼笔环肽染色检测破骨细胞肌动蛋白环，采用骨板吸收实验检测破骨细胞噬骨功能，采用pH计检测体系pH值，采用Western Blot检测破骨细胞分化相关蛋白表达。此外，提取小鼠背根神经节来源神经细胞并用淫羊藿苷处理，采用C...     

淫羊藿苷调节细胞增殖作用的研究进展

淫羊藿苷是传统补益中药淫羊藿的有效活性成分之一,研究发现可影响多种细胞的增殖。淫羊藿苷主要促进骨组织相关细胞增殖,包括成骨细胞、软骨细胞、骨膜细胞、间充质干细胞,淫羊藿苷抑制多种肿瘤细胞增殖,包括骨肉瘤细胞、乳腺癌细胞和卵巢癌细胞等,淫羊藿苷亦可调节其他细胞的增殖,包括神经干细胞、平滑肌细胞等。但淫羊藿苷对细胞增殖促进和抑制的双重调节机制需要进一步研究,方能为其在细胞增殖紊乱相关疾病中的应用提供理论依据。     

Icariin stimulates MC3T3-E1 cell proliferation and differentiation through up-regulation of bone morphogenetic protein-2

Previous studies suggest that icariin has anabolic effects on bone, but the mechanisms are unknown. We aimed to investigate the osteogenic effects of icariin in an undifferentiated osteoblast cell line by detecting cell morphology, viability, cell cycling and bone morphogenetic protein-2 (BMP-2) expression. We treated pre-osteoblastic MC3T3-E1 cells with different concentrations of icariin [0 (as a control), 10, 20 and 40 ng/ml] for 48, 72 and 96 h. Cell morphology, viability and the cell cycle were examined and measured using microscopy, the MTT assay or flow cytometry, respectively. BMP-2-positive cells and BMP-2 protein expression levels in icariin-treated MC3T3-E1 cells were examined using immunohistochemistry staining with fluorescence optical density analysis and Western blotting. MC3T3-E1 cells showed typical characteristics of osteoblasts in response to treatment with icariin. Cells treated with all concentrations of icariin had increased percentages of S-phase cells and decreased percentages of G1-phase cells, especially in the 10 and 20 ng/ml icariin groups. The number of BMP-2-positive cells and BMP-2 protein expression levels in the 10 and 20 ng/ml icariin treatment groups were greater compared to the 0 and 40 ng/ml groups. Treatment of icariin promotes osteoblast MC3T3-E1 proliferation and differentiation in vitro, potentially owing to its role in increasing BMP-2 protein expression. Icariin potentially can be used as a drug in clinical settings to treat osteoporosis.     

Differentiation of osteoblasts and in vitro bone formation from murine embryonic stem cells

Pluripotent embryonic stem (ES) cells have the potential to differentiate to all fetal and adult cell types and might represent a useful cell source for tissue engineering and repair. Here we show that differentiation of ES cells toward the osteoblast lineage can be enhanced by supplementing serum-containing media with ascorbic acid, beta-glycerophosphate, and/or dexamethasone/retinoic acid or by co-culture with fetal murine osteoblasts. ES cell differentiation into osteoblasts was characterized by the formation of discrete mineralized bone nodules that consisted of 50-100 cells within an extracellular matrix of collagen-1 and osteocalcin. Dexamethasone in combination with ascorbic acid and beta-glycerophosphate induced the greatest number of bone nodules and was dependent on time of stimulation with a sevenfold increase when added to ES cultures after, but not before, 14 days. Co-culture with fetal osteoblasts also provided a potent stimulus for osteogenic differentiation inducing a fivefold increase in nodule number relative to ES cells cultured alone. These data demonstrate the application of a quantitative assay for the derivation of osteoblast lineage progenitors from pluripotent ES cells. This could be applied to obtain purified osteoblasts to analyze mechanisms of osteogenesis and for use of ES cells in skeletal tissue repair.     

淫羊藿苷促进间充质干细胞成骨分化：为治疗骨缺损提供一个良好的方向

BACKGROUND:Recent studies have shown that icariin is a good bone-inducing factor that can promote the osteogenic differentiation of mesenchymal stem cells, providing a new hope for the treatment of bone defects. OBJECTIVE:To review research achievements in pharmacological effects of icariin effects on bone tissue metabolism as well as its effect to promote osteogenic differentiation of mesenchymal stem cells. METHODS:The first author retrieved CNKI and PubMed databases for relevant Chinese and English literatures using keywords of “icariin, stem cell, osteogenesis”, respectively. Articles regarding icariin, stem cells, osteogenesis were included, and repetitive studies were excluded. Totally 754 articles were retrieved initially. In accordance with inclusion and exclusion criteria, 41 articles were included in result analysis. RESULTS AND CONCLUSION:As the main ingredient of Herba epimedii, icariin functions as a good osteogenetic growth factor to promote the osteogenic differentiation of bone marrow mesenchymal stem cells. In recent years, icariin has been shown to promote adipose-, umbilical cord-, and periodontal ligament tissue-derived mesenchymal stem cells to differentiate into osteoblasts. But such studies are less reported. Until now, mesenchymal stem cells still exhibit unsatisfactory osteogenic ability in in vivo experiments. Given this, osteogenetic growth factors contribute to the osteogenic differentiation of mesenchymal stem cells. Therefore, the use of icariin is expected to provide a good strategy for bone defect repair.     

极低频电磁场对大鼠成骨细胞的影响

极低频电磁场(Extremely low-frequency electromagnetic field,ELEF)特性可以影响成骨细胞的增殖与分化。为了寻找有效的刺激骨折愈合的方法,我们研制了一种可产生多种波形,波形频率、强度可调的极低频脉冲电磁场刺激仪(频率0~300 Hz,强度0~40 mT),使用该仪器研究了矩形、脉冲、三角及正弦等多种波形对新生大鼠颅骨成骨细胞的影响。结果表明,脉冲波与矩形波电磁场可以显著刺激细胞增殖(P<0.05),抑制细胞分化(P<0.05),而正弦波形可以刺激细胞分化(P<0.05),但却抑制细胞的增殖(P<0.05)。     

Effects of Remifentanil Preconditioning on Osteoblasts under Hypoxia-Reoxygenation Condition

Background: Ischemia-reperfusion of bone occurs in a variety of clinical conditions, such as orthopedic arthroplasty, plastic gnathoplasty, spinal surgery, and amputation. Usually, cellular models of hypoxia-reoxygenation reflect in vivo models of ischemia-reperfusion. With respect to hypoxia-reoxygenation conditions, the effects of remifentanil on osteogenesis have received little attention. Therefore, we investigated the effects of remifentanil on the proliferation and differentiation of osteoblasts during hypoxic-reoxygenation.Methods: After remifentanil (0.1, 1 ng/mL) preconditioning for 2 hours, human osteoblasts were cultured under 1% oxygen tension for 24 hours. Thereafter, the cells were reoxygenated for 12 hours at 37 °C. The naloxone groups were treated with naloxone for 30 minutes before remifentanil treatment. We measured cell viability via MTT assay. Osteoblast maturation was determined by assay of bone nodular mineralization. Quantitative PCR and western blot methods were used to determine BMP-2, osteocalcin, Akt, type I collagen, osterix, TGF-β1, HIF-1α, and RUNX2 expression levels.Results: Osteoblast viability and bone nodular mineralization by osteoblasts is recovered by remifentanil preconditioning from hypoxia-reoxygenation insult. During hypoxic-reoxygenation condition, remifentanil preconditioning induced the expression of BMP-2, osteocalcin, Akt, type I collagen, osterix, TGF-β1, HIF-1α, and RUNX2 in osteoblasts.Conclusions: Under hypoxia-reoxygenation conditions, remifentanil preconditioning enhanced the cell viability and maturation of osteoblasts, and stimulated the expression of proteins associated with osteoblast proliferation and differentiation of the osteoblast. Our results suggest that remifentanil may help in the treatment of bone stress injuries.     

依普拉芬在牵张成骨中的作用*

背景：有研究表明适宜浓度的依普拉芬可以促进鸡胚额骨成骨细胞增殖与分化,提高其碱性磷酸酶活性,增强成骨细胞矿化能力,促进骨形成。 目的：观察依普拉芬在用种植型牵张器对兔下颌牵张时成骨的影响。 方法：选择日本大耳白兔建立下颌骨牵张成骨动物模型,再用依普拉芬干预。于牵张后1 d,1,2,4周取材,进行血清学检查,并采用免疫组织化学方法观察转化生长因子β1在不同时间段的表达情况。 结果与结论：依普拉芬干预的模型兔在牵张后1 d,1,2,4周的骨小梁逐渐形成,成骨细胞逐渐增多,血清碱性磷酸酶活性和转化生长因子β1的表达均增高(P < 0.05)。结果证实依普拉芬在牵张成骨中可有效加速新骨的形成。     

In vitro osteogenesis assays: influence of the primary cell source on alkaline phosphatase activity and mineralization

In trabecular bone fracture repair in vivo, osteogenesis occurs through endochondral ossification under hypoxic conditions, or through woven bone deposition in the vicinity of blood vessels. In vitro osteogenesis assays are routinely used to test osteoblastic responses to drugs, hormones, and biomaterials for bone and cartilage repair applications. These cell culture models recapitulate events that occur in woven bone synthesis, and are carried out using primary osteoblasts, osteoblast precursors such as bone marrow-derived mesenchymal stromal cells (BMSCs), or various osteoblast cell lines. With time in culture, cell differentiation is typically assessed by examining levels of alkaline phosphatase activity (an early osteoblast marker) and by evaluating the assembly of a collagen (type I)-containing fibrillar extracellular matrix that mineralizes. In this review, we have made a comparative analysis of published osteogenic assays using calvarial cells, calvaria-derived cell lines, and bone marrow stromal cells. In all of these cell types, alkaline phosphatase activity shows similar progression over time using a variety of osteogenic and mineralizing media conditions; however, levels of alkaline phosphatase activity are not proportional to observed mineralization levels.     

中药促成骨细胞增殖和分化的机制与作用

背景：中药促进成骨细胞增殖和分化的效果已经得到肯定,其在细胞分子水平的药理机制研究取得了很大进展。 目的：对国内外应用中药诱导成骨细胞增殖和分化的药理机制研究进展作一综述。 方法：应用计算机检索CNKI,Pubmed和sciencedirect数据库中2001-01/2010-12关于中药促进成骨细胞增殖和分化机制研究的文章,在标题和摘要中以“中药,成骨细胞,增殖,分化,机制”或“Chinese herbs,osteoblast,proliferation,differentiation, mechanism”为检索词进行检索。初检得到176篇文献,最终选择30篇文献进行综述。 结果与结论：中药通过对成骨细胞基因、信号通路、细胞因子和OPG/RANK/RANKL系统的调控,以及类雌激素样作用促进成骨细胞增殖和分化,其作用机制明确,将中药引入骨组织工程领域具有良好的发展前景。 关键词：中药;成骨细胞;增殖;分化;机制;组织工程 doi:10.3969/j.issn.1673-8225.2012.07.037     

淫羊藿苷对过载损伤后成骨细胞凋亡和骨架的影响

目的研究淫羊藿苷对过载损伤后成骨细胞的凋亡和细胞骨架的影响。方法应用4点弯曲加载装置模拟过载损伤力学环境,建立过载损伤模型,依据力学加载前后是否加入药物干预,实验分为空白对照组、单纯给药组、单纯损伤组、预防给药组和损伤治疗组。应用细胞流式术进行细胞凋亡检测。应用特异性荧光染料分别标记微丝蛋白和细胞核,在激光共聚焦显微镜下观察细胞骨架的变化。结果与对照组比,过载损伤组的凋亡率最高,单纯给药组最低(P0.05);在过载处理组中,与单纯过载损伤组比,预防给药组的凋亡率最低(P0.05)。单纯过载损伤组的细胞皱缩变形,微丝排列紊乱,骨架排列疏松,轮廓模糊,骨架甚至出现破碎等现象。预防给药组细胞骨架形态变化不大,细胞核未见明显变化。结论淫羊藿苷可以在一定程度上抑制过载损伤后成骨细胞的凋亡,维持细胞骨架稳定。