补肾活血固齿方对小鼠颅顶前成骨细胞骨钙素合成的影响

目的观察补肾活血固齿方对小鼠颅顶前成骨细胞(MC3T3-E1细胞)骨钙素(OCN)合成的影响。方法用10%补肾活血固齿方含药血清、无药血清、胎牛血清分别培养MC3T3-E1细胞24 h、48 h、72 h,125Ⅰ放射免疫法检测3组细胞上清液中OCN的含量。结果含药血清组24、48、72 h OCN含量与无药血清组、胎牛血清组同期比较差异均有统计学意义(P0.05),含药血清组24、48、72 h OCN含量显著高于无药血清组、胎牛血清组同期。结论补肾活血固齿方可提高MC3T3-E1细胞分泌OCN的水平,促进该细胞分化为成骨细胞,加速骨的形成。     

补肾活血固齿方对MC3T3-E1细胞矿化结节形成影响的研究

目的观察补肾活血固齿方对小鼠颅顶前成骨细胞(MC3T3-E1细胞)矿化结节形成的影响,探讨该方剂促进成骨的作用机制。方法依照体表面积折算成等效剂量的补肾活血固齿方灌胃大鼠,制备含药血清(含药血清组);同等剂量的蒸馏水灌胃大鼠,制备无药血清(无药血清组);胎牛血清作为空白对照(胎牛血清组)。MC3T3-E1细胞分别于补肾活血固齿方含药血清、无药血清、胎牛血清中培养14 d,利用茜素红染色法观察各组矿化结节的形成情况并计数。结果 3组细胞在14 d时均形成矿化结节,含药血清组矿化结节形成数量明显多于无药血清组及胎牛血清组(P均<0.05),无药血清与胎牛血清组矿化结节形成数量比较差异无统计学意义(P>0.05)。结论补肾活血固齿方可促进MC3T3-E1细胞向成骨细胞分化成熟,通过促进牙周骨组织的再生而发挥其药理作用。     

补肾活血固齿方对小鼠颅顶前成骨细胞增殖活性及细胞周期影响的实验研究

目的:观察补肾活血固齿方对小鼠颅顶前成骨细胞(MC3T3-E1细胞)增殖活性及细胞周期变化的影响,探讨该方剂治疗牙周炎的作用机理。方法:MC3T3-E1细胞分别于5%、10%、15%、20%补肾活血固齿方含药血清,10%无药血清,10%胎牛血清中培养12、24、36、48、60、72 h,MTT法检测补肾活血固齿方对MC3T3-E1细胞增殖活性的影响。另外MC3T3-E1细胞分别于10%补肾活血固齿方含药血清、无药血清、胎牛血清中培养24、48、72 h,用流式细胞仪检测该方剂对MC3T3-E1细胞细胞周期的影响。结果:含药血清与胎牛血清、无药血清组相比,MC3T3-E1细胞增殖活性及细胞周期差异无显著性(P0.05)。结论:补肾活血固齿方对MC3T3-E1细胞增殖活性及细胞周期无影响,其可能是通过促进该细胞向成骨细胞分化、促进牙槽骨的再生而发挥药物作用。     

补肾活血固齿方对大鼠MC3T3-E1细胞BMP2表达及超微结构的影响

目的:研究补肾活血固齿方对小鼠颅顶前成骨细胞(MC3T3-E1细胞)BMP2表达及超微结构的影响,探讨该方剂治疗牙周炎的作用机理。方法:MC3T3-E1细胞分别于补肾活血固齿方含药血清、无药血清中培养24、48、72 h,ELISA法检测MC3T3-E1细胞BMP2的表达;培养14 d后,透射电镜观察MC3T3-E1细胞超微结构的变化。结果:含药血清组培养上清液的光密度值明显高于无药血清组,统计结果显示差异有显著性(P0.05)。MC3T3-E1细胞经药物作用后呈现成骨细胞样表型,细胞表面突起变多,核卵圆形、较大,核膜凹陷,核仁明显;胞质丰富,粗面内质网扩张明显,网腔内充满低电子密度絮状物;高尔基复合体、线粒体发达,具有良好的的分泌功能,细胞代谢活跃。结论:补肾活血固齿方能促进MC3T3-E1细胞分泌BMP2,参与诱导成骨作用;补肾活血固齿方能诱导MC3T3-E1细胞呈成骨细胞样表型,促进成骨细胞的分化生长。     

壮骨胶囊含药血清对小鼠成骨细胞增殖 及碱性磷酸酶、骨钙素合成的影响

目的观察壮骨胶囊含药血清对小鼠成骨细胞(MC3T3-E1细胞)增殖、碱性磷酸酶(ALP)、骨钙素(BGP)合成的影响,探讨壮骨胶囊对骨代谢、骨形成机制的作用。方法壮骨胶囊由射干、白芍等组成。实验分为对照组、壮骨胶囊高、中、低剂量组(4.3、2.15、1.08 g生药/kg),给药组灌胃给予Wistar大鼠壮骨胶囊,对照组给于等体积水,连续给予7 d后,采血离心制备对照组空白血清及含药组血清;以小鼠成骨细胞MC3T3-E1为研究对象,用空白血清及含药血清处理体外培养的MC3T3-E1成骨细胞,采用CCK8法检测成骨细胞增殖能力,IFCC速率法测定ALP活性,酶联免疫法测定BGP分泌量。结果壮骨胶囊含药血清各剂量组对体外培养的MC3T3-E1细胞增殖有显著的促进作用,高、中剂量组48 h可促进成骨细胞增殖(同对照组比较,P 0.05),高、中、低剂量组72 h均能促进成骨细胞增殖(同对照组比较,P 0.01,P 0.05);高、中剂量组含药血清作用于MC3T3-E1细胞72 h后,ALP活性、BGP分泌量较对照组明显提高(P 0.01,P 0.05)。结论壮骨胶囊含药血清具有促进骨形成作用,这可能与其促进成骨细胞增殖,增加成骨细胞ALP的合成和BGP的分泌,从而改善骨代谢有关。     

补肾活血中药对体外培养成骨细胞活性的影响

目的研究补肾活血中药对体外培养成骨细胞活性的影响.方法取二次酶消化法培养的胎鼠颅骨第三代成骨细胞,随机分为5组,即:细胞对照组、空白血清组、含药血清组、原药组、阳性对照药α D3组,分别加入普通细胞培养液、含空白血清及不同浓度补肾活血中药含药血清、补肾活血中药原药液、阳性对照药(αD3)的条件培养液,分别在培养第3天和第6天进行各试验组成骨细胞碱性磷酸酶及骨钙蛋白分泌量测定,观察补肾活血中药含药血清及原药液对成骨细胞活性的影响.结果含药血清组成骨细胞活性指标碱性磷酸酶(ALP)值在培养第3天均低于空白血清组,且与含药血清浓度呈负相关关系;第6天,含药血清组各浓度组ALP值明显升高,均显著高于空白血清组,ALP值与含药血清浓度存在正相关关系;空白血清组ALP值在培养第6天时下降,其均值低于培养第3天ALP值.培养第3天及第6天,各浓度原药组ALP值均高于细胞对照组及αD3组;ALP值与原药浓度呈正相关关系.含药血清组及中药原药组骨钙素(OC)含量显著高于空白血清及其他各对照组.结论补肾活血中药含药血清及原药能够直接作用于体外培养的成骨细胞,促进成骨细胞增殖,增强其分泌活性;可延长成骨细胞活性分泌期,使其活性分泌高峰后移而峰值升高,增加其活性分泌总量.     

基于促MC3T3-E1细胞增殖与分化的不同规格鹿茸聚类分析

目的考察8种不同规格鹿茸含药血清对MC3T3-E1成骨细胞增殖和分化的影响并对8种不同规格鹿茸进行聚类分析。方法雌性SD大鼠随机分为9组,给药,制得不同规格鹿茸的大鼠含药血清及空白对照血清。以上述血清对MC3T3-E1成骨细胞培养72 h,MTT法检测成骨细胞增殖情况,ELISA法检测碱性磷酸酶(ALP)分泌量,并以成骨细胞的增殖及ALP分泌量为因素进行聚类分析。结果各规格鹿茸均具有促进MC3T3-E1成骨细胞增殖、分化作用,且聚类分析结果与传统商品鹿茸药材质量划分结果相同。结论鹿茸药材能显著促进MC3T3-E1成骨细胞增殖、分化,且作用强弱与商品药材优劣相关。     

六味地黄丸含药血清对MC3T3-E1细胞增殖以及对Runx2、FOXO1 mRNA表达的影响

目的探讨六味地黄丸含药血清对MC3T3-E1细胞增殖以及对Runx2、FOXO1 mRNA表达的影响。方法制作六味地黄丸混悬液,对成年Wistar大鼠进行灌胃,获取六味地黄丸含药血清。常规培养MC3T3-E1细胞24 h后更换含10%不同浓度含药血清的培养基,分别培养48 h和72 h后用CCK-8法检测MC3T3-E1细胞的增殖;对MC3T3-E1细胞进行诱导培养22天后进行饥饿培养24h,随后更换含10%不同浓度的含药血清,培养72 h后检测Runx2、FOXO1 mRNA表达。结果六味地黄丸含药血清能促进MC3T3-E1细胞增殖,并且呈现一定的剂量依赖性;同时六味地黄丸含药血清各剂量组均能明显促进Runx2 mRNA的表达(P0.01),高剂量组的表达明显高于低剂量组(P0.01)。高剂量组的FOX01 mRNA表达明显高于其他3组(P0.01)。结论六味地黄丸含药血清能促进MC3T3-E1细胞增殖,同时能促进Runx2、FOXO1 mRNA表达。     

六味地黄方含药血清对H2O2诱导的氧化损伤MC3T3-E1细胞的干预作用

目的 观察六味地黄方含药血清对H₂O₂诱导的氧化损伤小鼠胚胎成骨细胞前体细胞(MC3T3-E1)增殖、分化、矿化的干预作用,并从无翅型MMTV整合位点家族成员/β-连环蛋白(Wnt/β-Catenin)、护骨素/核因子-κB受体活化因子配体(OPG/RANKL)通路及线粒体凋亡途径初步探讨其作用机制。方法 制作六味地黄方汤剂,对成年Wistar大鼠进行灌胃,获取六味地黄方含药血清。将MC3T3-E1细胞分为正常组、模型组、N-乙酰半胱氨酸(NAC)组、对照含药血清组、低剂量含药血清组、中剂量含药血清组及高剂量含药血清组。除正常组外,其余各组先用1.0 mmol/L过氧化氢(H₂O₂)预处理MC3T3-E1细胞6 h,随后模型组更换为正常培养基,NAC组更换为含有2.5 mmol/L NAC的培养基,药物血清组更换为含有10%药物血清的培养基,各组均处理24 h,然后根据检测指标的不同给予进一步处理。采用CCK-8法检测培养24 h、48 h、72 h后MC3T3-E1细胞的增殖情况,诱导培养7 d后...     

二仙汤及其拆方含药血清对成骨细胞分化的影响

目的:研究二仙汤及其君、臣、佐、使拆方含药血清对成骨细胞分化的影响。方法:实验以成骨细胞系MC3T3-E1细胞为基础,分为空白组、白藜芦醇15mg/kg组、二仙汤整方8g/kg组及依据君臣佐使关系的4个拆方组,即淫羊藿+仙茅3.2g/kg组、淫羊藿+仙茅+巴戟天4.8g/kg组、淫羊藿+仙茅+当归4.8g/kg组、淫羊藿+仙茅+知母+黄柏4.8g/kg组。分别灌胃大鼠,制备含药血清,分别以10%含药血清作用于成骨细胞,以对硝基苯磷酸盐(pNPP)法和碱性磷酸酶(ALP)染色检测成骨细胞ALP活性;以Von Kossa染色法检测成骨细胞矿化结节形成;采用Western blot法检测成骨细胞骨钙素(OC)、骨形成蛋白-2(BMP-2)的表达。结果:与空白组比较,二仙汤整方和各拆方组10%含药血清均能显著增强成骨细胞ALP的活性,其中二仙汤整方组、淫羊藿+仙茅+知母+黄柏组提高成骨细胞ALP活性的作用最为明显,二仙汤整方和各拆方组均可显著提高成骨细胞OC、BMP-2的表达。结论:二仙汤及各拆方含药血清均不同程度地提高成骨细胞ALP活性,增强矿化结节的形成,促进OC、BMP-2的表达,知母、黄柏的作用尤为重要。     

竹节参对MC3T3-E1成骨样细胞增殖与分化的影响

目的：研究竹节参不同提取物对体外培养成骨样细胞（MC3T3-E1）增殖、分化作用的影响。方法：采用MC3T3-E1细胞为体外药物筛选的细胞模型,用MTT法测定药物对成骨细胞的增殖作用,碱性磷酸酶（ALP）试剂盒测定ALP的活性,考察竹节参不同提取物对MC3T3-E1细胞增殖、分化作用的影响。结果：10^-1mg／mL的竹节参水提物和10^-4mg／mL的95％乙醇提取物能显著促进MC3T3-E1细胞的增殖和分化（P〈0．05）。结论：一定浓度的竹节参水提物和95％乙醇提取物能显著促进MC3T3-E1细胞的增殖和分化,该药物具有开发抗骨质疏松药物的潜力。     

基于分子对接的方法探讨桑根酮C对MC3T3-E1细胞的作用

目的:观察桑根酮C(SanC)对地塞米松(DEX)作用下小鼠MC3T3-E1成骨细胞增殖与分化的影响,并探讨其作用机制。方法:将SanC与同源建模所得的Runt-相关转录因子2(Runx2)蛋白结构进行分子对接。不同浓度SanC(8,16,32μmol·L^-1)和1μmol·L^-1DEX共同作用MC3T3-E1细胞,而后采用细胞增殖-毒性检测试剂盒(CCK-8)法检测SanC对MC3T3-E1成骨细胞增殖影响。试剂盒测定MC3T3-E1成骨细胞碱性磷酸酶(ALP)活性和茜素红染色检测骨矿化结节的形成。采用实时荧光定量聚合酶链反应(Real-time PCR)检测Runt-相关转录因子2(Runx2),ALP,和锌指结构转录因子(Osterix)mRNA的表达水平。蛋白免疫印迹法(Western blot)检测Runx2蛋白表达。结果:SanC与Runx2对接打分为-9.78。与正常组比较,DEX组显著降低细胞存活率(P<0.01),其中7 d存活率差异达到最大;与DEX组比较,SanC能显著促进MC3T3-E1的细胞增值(P<0.01),其中32μmol·L^-1SanC作用细胞7 d增殖率差异达到最大。与正常组比较,DEX组Runx2,ALP和Osterix mRNA的表达均有一定程度升高(P<0.05);与DEX组比较,不同浓度SanC组依赖性上调Runx2,ALP和Osterix mRNA的表达(P<0.01)。与正常组比较,DEX组Runx2蛋白表达明显下降(P<0.05);与DEX组比较,SanC干预下细胞Runx2蛋白表达显著升高(P<0.01)。结论:桑根酮C能促进MC3T3-E1成骨细胞增殖、分化和矿化,其机制可能与上调Runx2表达有关。     

仙茅苷促MC3T3-E1成骨样细胞增殖、分化及钙化作用的研究

目的:考察仙茅苷对MC3T3-E1成骨样细胞的增殖、分化及钙化功能的影响。方法:用不同浓度仙茅苷加入MC3T3-E1细胞培养体系中,MTT法检测细胞增殖水平;用茜素红染色法考察骨小结形成能力;以对硝基苯二钠基质动力学法检测碱性磷酸酶的活性。结果:仙茅苷(10-4～10-8mol.L-1)对细胞增殖有促进作用,高浓度(10-4～10-6 mol.L-1时作用明显,其中48h为最佳作用时间;仙茅苷以10-7、10-9 mol.L-1浓度在96h时可促进MC3T3-E1细胞碱性磷酸酶活性;仙茅苷浓度为10-9 mol.L-1时对于骨小结形成最为有效。结论:仙茅苷对MC3T3-E1成骨样细胞的增殖、分化及骨小结形成均有促进作用。     

Effect of safflower seeds supplementation on stimulation of the proliferation, differentiation and mineralization of osteoblastic MC3T3-E1 cells

Anti-bone resorption properties of the Korean herbal formulation, Gami-Honghwain (HJ), which comprises Carthamus tinctorius L. seed and hominis placenta, were investigated. We demonstrate that the production of PGE2 is inhibited by 20-100 microg/ml HJ in nontransformed osteoblastic cells (MC3T3-E1 cells), indicating that HJ inhibits PGE2 production. The effect of HJ on the proliferation and osteoblastic differentiation in MC3T3-E1 was also studied. HJ dose-dependently increased DNA synthesis (significant at 20-100 microg/ml), and increased alkaline phosphatase (ALP) and prolyl hydroxylase activities of MC3T3-E1 cells (20-100 microg/ml), while anti-estrogen tamoxifen eliminated the stimulation of proliferation and ALP activity of MC3T3-E1 which was induced by HJ. These results indicate that HJ directly stimulates cell proliferation and differentiation of osteoblasts. Also, when we assessed the effects of HJ on osteoblastic differentiation in MC3T3-E1, HJ enhanced ALP activity and mineralization in a dose- and time-dependent fashion. This stimulatory effect of the HJ was observed at relatively low doses (significant at 20-100 microg/ml and maximal at 100 microg/ml). Northern blot analysis showed that the HJ (60 microg/ml) increased in bone morphogenetic protein-2 as well as ALP mRNA concentrations in MC3T3-E1 cells. HJ (100 microg/ml) slightly increased in type I collagen mRNA abundance throughout the culture period, whereas it markedly inhibited the gene expression of collagenase-1 between days 15 and 20 of culture. These results indicate that HJ has anabolic effect on bone through the promotion of osteoblastic differentiation, suggesting that it could be used for the treatment of common metabolic bone diseases.     

下瘀血汤抗猪血清免疫性肝纤维化方证相关的药效学研究

目的：研究下瘀血汤抗大鼠猪血清肝纤维化方-证相关的药效学机制。方法：猪血清腹腔注射复制肝纤维化模型,造模8周后继续造模的同时以下瘀血汤、茵陈蒿汤、黄芪汤、二至丸和小柴胡汤进行干预治疗。测定大鼠血清ALT、AST、ALP和GGT活性,Alb、TP和TBil含量;Jamall法测定肝组织羟脯氨酸（Hyp）含量。结果：与正常组比较,模型组大鼠血清ALT和AST活性增高、Alb和TP含量显著下降（P〈0.05）,ALP和GGT活性及TBiL含量虽有升高但无显著性差异（P〉0.05）;与模型组比较,下瘀血汤、茵陈蒿汤和小柴胡汤组大鼠血清肝功能有一定的改善,以下瘀血汤组为最佳;与正常组比较,模型组大鼠肝组织Hyp含量显著增加（P〈0.05）;与模型组比较,下瘀血汤组大鼠肝组织Hyp含量显著降低（P〈0.05）,其它各药物组无改善作用（P〉0.05）。结论：下瘀血汤可有效的抑制大鼠猪血清免疫性肝纤维化,提示本模型病理阶段的证候为瘀血阻络。     

Stimulative effects of Drynariae Rhizoma extracts on the proliferation and differentiation of osteoblastic MC3T3-E1 cells

Pharmacological factors are needed to prevent bone loss that occurs with increasing age. The chemical compounds that act on bone metabolism in herbal medicines, however, are poorly understood. Effects of traditional Korean medicine, Drynariae Rhizoma [Drynaria fortunei (kunze) J. Sm] extract (DR), on the osteoblastic proliferation and differentiation were investigated. The effect of DR, a natural phyto herb, on the proliferation and osteoblastic differentiation in non-transformed osteoblastic cells (MC3T3-E1) was studied. DR dose-dependently increased DNA synthesis (significant at 50-150 microg/ml). DR increased alkaline phosphatase (ALP) activity and prolyl hydroxylase activity of MC3T3-E1 cells (50-150 microg/ml). Antiestrogen tamoxifen eleminated the stimulation of proliferation and ALP activity of MC3T3-E1, which were induced by DR. DR at concentrations ranged from 30-100 microg/ml inhibited prostaglandin E2 production in MC3T3-E1. These results indicate that DR directly stimulates cell proliferation and differentiation of osteoblasts. These results also suggest and DR is effective for bone anti-resorptive action in bone cells.     

Stimulative effects of Ulmus davidiana Planch (Ulmaceae) on osteoblastic MC3T3-E1 cells

Ulmus davidiana Planch (Ulmaceae) has long been known to have anti-inflammatory and protective effects on damaged tissue, inflammation and bone among other functions. To treat rheumatoid arthritis (RA), a herbal medicine, Ulmus davidiana Planch (Ulmaceae) extract (UD) is being used in traditional oriental medicine. The effect of UD on the proliferation and osteoblastic differentiation in non-transformed osteoblastic cells (MC3T3-E1) was studied. UD dose-dependently increased DNA synthesis (significant at 5-20 microg/ml). UD increased alkaline phosphatase (ALP) activity and prolyl hydroxylase activity of MC3T3-E1 cells (5-20 microg/ml). Antiestrogen tamoxifen eliminated the stimulation of proliferation and ALP activity of MC3T3-E1, which was induced by UD. UD at concentrations ranged from 30 to 100 microg/ml inhibited prostaglandin E2 production in MC3T3-E1. These results indicate that UD directly stimulates cell proliferation and differentiation of osteoblasts. These results also suggest and UD is effective for bone anti-resorptive action in bone cells.     

Stimulatory effects of extract prepared from the bark of Cinnamomum cassia blume on the function of osteoblastic MC3T3-E1 cells

The ethanol extract from the bark of Cinnamomum cassia Blume (CCE) was tested for estrogenic activity. CCE (4-60 microg/mL) significantly induced the growth of MCF-7 cells, an ER-positive human breast cancer cell line, over that of untreated control cells (p < 0.05). In the ER competitive binding assay, CCE showed higher affinity with ERbeta compared with ERalpha. To investigate the bioactivities of CCE, which act on bone metabolism, the effects of CCE on the function of osteoblastic MC3T3-E1 cells and the production of local factors in osteoblasts were studied. CCE (4-60 microg/mL) dose-dependently increased the survival of MC3T3-E1 cells. In addition, CCE (10 and 50 microg/mL) increased alkaline phosphatase (ALP) activity, collagen synthesis and osteocalcin secretion in MC3T3-E1 cells. Treatment with CCE (10 and 50 microg/mL) prevented apoptosis induced by TNF-alpha (10(-10) m) in osteoblastic cells. In the presence of TNF-alpha, culture with CCE (10-100 microg/mL) for 48 h inhibited the production of IL-6 and nitric oxide in osteoblastic MC3T3-E1 cells. These results suggest that Cinnamomum cassia has a direct stimulatory effect on bone formation in vitro and may contribute to the prevention of osteoporosis and inflammatory bone diseases.