金叶子异槲皮苷对MC3T3-E1细胞成骨分化的影响

该研究对从金叶子中分离得到的异槲皮苷的成骨活性进行了系统评价。在1×10-4,1×10-5,1×10-6,1×10-7mol·L-1异槲皮苷浓度作用下检测了MC3T3-E1细胞增殖活力和碱性磷酸酶活性;在异槲皮苷作用的第3天对MC3T3-E1碱性磷酸酶、I型胶原以及转录因子Runx2和Osterix的基因表达水平进行了检测;并通过茜素红染色的方法在第21天对MC3T3-E1进行了胞外基质矿化能力的评价。结果显示,异槲皮苷在1×10-7~1×10-5mol·L-1能促进MC3T3-E1细胞的增殖、分化及矿化能力,上调成骨相关基因的表达,而且该作用呈现出一定的浓度依赖性,在浓度为1×10-6mol·L-1时促进作用最强。在1×10-4mol·L-1时表现出明显的细胞毒性。因此,从金叶子中分离得到的异槲皮苷有一定的成骨活性,这可能是传统中药金叶子治疗骨折的主要药效成分。     

黄芪甲苷对MC3T3-E1细胞活性的影响及其机制探讨

目的:探讨黄芪甲苷对小鼠胚胎成骨细胞MC3T3-E1细胞活性的影响及其作用机制。方法:体外培养MC3T3-E1细胞,用不同浓度的黄芪甲苷进行预处理后,MTT法测定细胞增殖情况,碱性磷酸酶法(ALP)测定细胞分化情况,Western blotting分析转化生长因子TGF-β1、成骨细胞信号转导蛋白Smad2/3表达水平;应用小干扰RNA(siRNA)转染法观察TGF-β1 siRNA对MC3T3-E1细胞增殖及活性的影响,加入抗Smad2/3抗体,探讨Smad2/3在黄芪甲苷介导的细胞增殖分化中的作用。结果:黄芪甲苷在一定浓度范围内剂量依赖性促进MC3T3-E1细胞的增殖和分化能力。进一步机制分析表明,黄芪甲苷处理可诱导TGF-β1蛋白表达,TGF-β1siRNA处理后,黄芪甲苷诱导的MC3T3-E1细胞的增殖和分化能力明显下降;此外,黄芪甲苷可显著性诱导Smad2/3表达,经TGF-β1 siRNA处理后,黄芪甲苷诱导的Smad2/3表达显著下降;加入Smad2/3抗体后,黄芪甲苷诱导的MC3T3-E1细胞的增殖和分化能力明显下降。结论:黄芪甲苷可通过刺激TGF-β1-Smad2/3通路的活化促进成骨细胞的增殖和分化,从而有利于骨形成。因此,本研究将为骨折愈合的治疗提供新的研究方向。     

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。     

柚皮苷对小鼠成骨细胞MC3T3 E1增殖、分化和矿化的影响

目的:检测骨碎补有效成分柚皮苷对成骨细胞株MC3T3-E1细胞增殖、分化和矿化的影响.方法:以成骨细胞株MC3T3-E1细胞为体外药效的试验模型(药物组和对照组),通过CCK-8法观察10,1,0.1,0.01,0.001,0.000 1μmol·L~(-1)的柚皮苷溶液对MC3T3-E1细胞增殖能力的影响;通过乳酸脱氢酶(LDH)细胞毒性试验观察以上浓度的柚皮苷溶液对细胞毒性能力的影响.通过骨形成蛋白-2(BMP-2),碱性磷酸酶(ALP)活性测定和骨钙素(OC)含量测定观察柚皮苷对MC3T3-E1细胞分化能力的影响;通过Von kossa钙化染色法观察柚皮苷对MC3T3-E1细胞钙化能力的影响.结果:高浓度的柚皮苷溶液在12 h和24 h时能促进MC3T3-E1细胞的增殖作用.而低浓度的柚皮苷溶液则无此作用.所测的各个组别细胞毒性百分比都较小,且随着时间的推移(12,24,48 h)变化较小.光镜下观察各个时间点细胞的密度和形态也未发生明显的变化.BMP-2细胞免疫化学结果表明,24,48 h时柚皮苷浓度10,1,0.1μmol·L~(-1)包浆内棕色着色比对照组明显.ALP检测结果表明48 h时,1,0.1μmol·L~(-1)的柚皮苷溶液可提高MC3T3细胞的ALP活性(P<0.05).72 h时,0.1 μmol·L~(-1)的柚皮苷溶液可提高MC3T3细胞的ALP活性(P<0.05).OC检测结果表明12 d时,10,1μmol·L~(-1)柚皮苷溶液作用组与对照组相比OC活性明显提高(P<0.05).Vonkossa染色钙化面积百分比结果表明3组柚皮苷溶液作用组与对照组相比无明显区别.结论:柚皮苷溶液可以提高成骨细胞株MC3T3-E1增殖能力和分化能力.     

淫羊藿总黄酮胶囊活性成分宝藿苷Ⅱ对MC3T3-E1细胞增殖分化的影响

目的:从淫羊藿总黄酮胶囊中制备分离宝藿苷Ⅱ,检测不同浓度宝藿苷II对体外培养的MC3T3-E1细胞增殖分化的影响。方法:分别用10、20、40nmol/ml的宝藿苷II作用MC3T3-E1细胞,CCK-8法检测细胞存活率;比色法测定碱性磷酸酶(ALP)活力;ELISA法检测骨钙素(OT)含量;茜素红染色分析并用氯化十六烷基吡啶测定钙沉积量;Western Blot法检测宝藿苷Ⅱ对BMP2信号通路蛋白表达的影响。结果:10、20、40nmol/ml的宝藿苷Ⅱ均可明显促进MC3T3-E1的增殖;提高MC3T3-E1分泌ALP和OT的能力;矿化结节数量明显增多且钙沉积量吸光度值显著增大;明显提高BMP2、Runx2和Osterix蛋白表达量,且呈剂量依赖性。结论:淫羊藿总黄酮胶囊活性成分宝藿苷II可以明显促进MC3T3-E1细胞的增殖分化。     

8味中药提取物对MC3T3-E1细胞增殖及碱性磷酸酶活性的影响

目的:考察中药沙苑子、续断、桑寄生、鸡血藤、甘草、巴戟天、葫芦巴、射干不同提取部位对MC3T3-E1细胞增殖及碱性磷酸酶活性的影响.方法:将8味中药的醇提及不M极性溶剂萃取物与MC3T3-E1细胞体外培养,用MTT法榆测各部位对成骨细胞MC3T3-E1的增殖影响;用PNPP法测定对成骨细胞MC3T3-E1细胞内和细胞外碱性磷酸酶活性的影响.结果:MC3T3-E1细胞经实验组药物和10~(-7)mol/L雌酚酮处理后96 h,与卒自对照组相比,增殖速度显著加快的有:桑寄生的氯仿和乙酸乙酯萃取物、葫芦巴萃余物、射干的乙酸乙酯萃取物;细胞内ALP活性增加与对照组相比有显著性差异(P<0.01)的有:桑寄生、鸡血藤、巴戟大萃余物,细胞外ALP活性增加与对照组相比有显著性差异(P<0.01)的有:鸡血藤、巴戟天、射干的萃余物和葫芦巴石油醚萃取物.结论:鸡血藤、桑寄生、巴戟天、葫芦巴和射干具有促进成骨细胞合成和分泌ALP的作用,提示其具有促进成骨细胞增殖和分化的作用,可进一步研究其体内的作用,可为开发抗骨质疏松药物.     

淫羊藿苷促前成骨细胞MC3T3-E1成骨分化的实验研究

目的探讨淫羊藿苷(icariin,ICA)促进前成骨细胞MC3T3-E1成骨分化的机制。方法采用不同浓度(10~(-10)~10~(-5)mol/L)的ICA干预前成骨细胞MC3T3-E1 3、6、9天后采用碱性磷酸酶(alkaline phosphatase,ALP)活性检测试剂盒测定ALP活性,确定ICA最适促分化浓度。最适ICA浓度干预前成骨细胞MC3T3-E1 7天后应用实时定量PCR法(real time-polymerase chain reaction,RT-PCR)检测骨形态发生蛋白(BMP)和Wnt/β-catenin信号通路相关分子(BMP-2、Runx2、β-catenin、cyclin D1)基因表达。ICA分别与BMP、Wnt/β-catenin信号通路抑制剂(Noggin、DKK-1)联合干预前成骨细胞MC3T3-E1后,茜素红钙结节染色观察钙化程度以及Western blot检测BMP-2蛋白表达变化。结果 ICA促进前成骨细胞MC3T3-E1成骨分化,且10~(-9)mol/L ICA促进分化的能力最强。与对照组比较,ICA能明显上调BMP和Wnt/β-catenin信号通路相关分子(BMP-2、Runx2、β-catenin、cyclin D1)mRNA表达(P0.01)。Noggin或DKK-1能抑制前成骨细胞MC3T3-E1钙化,且两者联合作用时抑制更加明显。与ICA单独作用比较,ICA与DKK1联合干预后BMP-2蛋白表达水平下降(P0.01)。结论 ICA可能通过Wnt/β-catenin/BMP-2信号通路调控前成骨细胞MC3T3-E1的成骨分化。     

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。     

新疆马鹿角提取物对成骨细胞MC3T3-E1增殖、分化和矿化的影响

目的 研究新疆马鹿角提取物对体外培养成骨细胞株MC3T3-E1细胞增殖、分化和矿化的影响.方法 以MC3T3-E1细胞作为药物筛选模型,用MTT法测定不同浓度的新疆马鹿角提取物促细胞增殖作用,采用碱性磷酸酶活性测定和骨钙素含有量检测,观察不同浓度的新疆马鹿角提取物促细胞分化作用,以Von kossa钙化染色法观察新疆马鹿角提取物溶液对MC3T3-E1细胞钙化能力的影响.结果 MTT检测结果表明5×102、5×101、5 μg/mL新疆马鹿角提取物溶液在24、48、72 h均可促进MC3T3-E1细胞增殖,且呈时间依赖性.ALP检测结果表明5×102、5×101 μg/mL新疆马鹿角提取物溶液在24、48、72 h可提高MC3T3-E1细胞内ALP的活性.BGP检测结果表明5×102、5×101 μg/mL新疆马鹿角提取物溶液在8、12 d可促进MC3T3-E1细胞内BGP的合成和分泌.Von kossa染色钙化面积百分比结果表明3组新疆马鹿角提取物溶液与对照组比较无明显区别.结论 新疆马鹿角提取物可促进成骨细胞株MC3T3-E1的增殖和分化能力.     

白芍总苷对小鼠成骨细胞增殖、分化和矿化的影响

目的研究白芍总苷(TGP)对小鼠成骨细胞(MC3T3-E1)增殖、分化和矿化的影响。方法采用MTT法和流式细胞术检测TGP对MC3T3-E1细胞增殖和细胞周期的影响;诱导MC3T3-E1成骨分化,通过检测碱性磷酸酶(ALP)活性和RT-PCR法检测人类相关转录因子2(Runx2)基因表达,分析TGP对成骨分化的影响,采用茜素红染色分析TGP对矿化的影响。结果 3、10μg/L TGP均能促进MC3T3-E1细胞增殖(P0.05,P0.01);10μg/L TGP可使G1期细胞的比例减少,S+G2期细胞的比例增加(P0.05);与对照组比较,TGP处理组细胞的ALP活性和Runx2表达水平显著升高(P0.05,P0.01),且矿化结节量明显增加。结论 TGP具有促进MC3T3-E1细胞增殖和分化成熟的能力,为炎性骨质疏松的治疗策略提供了实验依据和治疗思路。     

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.     

骨碎补提取物促小鼠成骨细胞株MC3T3-E1细胞增殖、分化和钙化作用的研究

目的 :探讨中药骨碎补有无促进成骨细胞增殖、分化和钙化的作用。方法 :制备骨碎补的水、醇提取液。小鼠成骨细胞株MC3T3 E1作为药物筛选的细胞模型 ;用MTT法和流式细胞术测定不同浓度的骨碎补水、醇提取液的促细胞增殖作用 ;采用ALP活性和骨钙素定量检测分别观察上述药物提取液的促细胞分化作用 ;以Vonkossa钙化染色法了解上述药物提取液的促细胞钙化作用。结果 :0.01,1mg·L^-1骨碎补水提液能促进MC3T3 E1细胞数量增加 (P <0 .0 5 ) ;0.01,1mg·L^-1骨碎补水提液和醇提液能使S期细胞百分率升高、G1期细胞百分率减少 ;1,10 0mg·L^-1骨碎补醇提液能使细胞ALP的活性升高 (P <0.05 ) ;100mg·L^-1骨碎补醇提液能明显促进细胞骨钙素合成和分泌 (P <0 .001) ;1mg·L^-1骨碎补水提液及 0.01mg·L^-1骨碎补醇提液均可促进细胞钙化 (P <0.05 )。结论 :骨碎补水相和醇相提取物中分别存在有较高活性的促成骨细胞增殖、分化和钙化的物质。     

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.     

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.     

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.     

补肾活血固齿方对小鼠颅顶前成骨细胞碱性磷酸酶活性的影响

目的观察补肾活血固齿方对小鼠颅顶前成骨细胞(MC3T3-E1)碱性磷酸酶(ALP)活性的影响,探讨其对成骨细胞分化的作用机制。方法将20只SD大鼠随机分为含药血清组和无药血清组,各10只。含药血清组大鼠予补肾活血固齿方灌胃,无药血清组予等容积0.9%氯化钠注射液灌胃,均灌胃7 d后抽取腹主动脉血制备成含药血清和无药血清。胎牛血清组为直接购买的PAA胎牛血清。分别用含10%含药血清、10%无药血清及10%胎牛血清的培养基培养MC3T3-E1细胞24、48、72 h,检测MC3T3-E1细胞中ALP的含量。结果含药血清组培养24、48、72 h后MC3T3-E1细胞ALP含量均高于胎牛血清组及无药血清组同期水平,比较差异均有统计学意义(P0.05),但胎牛血清组与无药血清组同期差异无统计学意义(P0.05);含药血清组MC3T3-E1细胞ALP含量随培养时间的延长而明显增加(P0.05)。结论补肾活血固齿方可增强MC3T3-E1细胞中ALP活性,促进MC3T3-E1细胞向成骨细胞分化,且均有一定时间依赖性。