龙牡壮骨颗粒对小鼠成骨细胞MC3T3-E1增殖、分化及矿化的影响

目的:研究龙牡壮骨颗粒对小鼠成骨细胞株MC3T3-E1增殖、分化和矿化的影响。方法:以小鼠成骨细胞株MC3T3-E1作为药物筛选的体外模型;采用终浓度分别为1,5,10,25,50μg/ml的龙牡壮骨颗粒溶液干预,MTT法检测细胞增殖率;同时检测药物作用不同时间后的碱性磷酸酶(ALP)活性;茜素红染色和Von Kossa钙化染色检测不同浓度药物对细胞钙化的影响。结果:龙牡壮骨颗粒溶液在5⁵0μg/ml范围内培养24和48 h可显著促进小鼠成骨细胞增殖;龙牡壮骨颗粒溶液在550μg/ml范围内培养24和48 h可显著促进小鼠成骨细胞增殖;龙牡壮骨颗粒溶液在5⁵0μg/ml范围内培养48和72 h显著提高MC3T3-E1细胞ALP活性。龙牡壮骨颗粒浓度为10μg/ml和50μg/ml时,茜素红染色钙化结节显著增多,药物浓度在1050μg/ml范围内培养48和72 h显著提高MC3T3-E1细胞ALP活性。龙牡壮骨颗粒浓度为10μg/ml和50μg/ml时,茜素红染色钙化结节显著增多,药物浓度在10⁵0μg/ml范围内,Von Kossa染色钙化结节显著增多。结论:龙牡壮骨颗粒可提高MC3T3-E1增殖、分化及矿化的能力。     

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.     

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.     

酢浆草提取物对成骨细胞增殖及分化的影响

目的:探讨酢浆草水提物对体外培养的成骨细胞增殖、分化和矿化的影响。方法:不同质量浓度酢浆草水提物(10,100,200 mg·L-1)作用人成骨肉瘤细胞株SaOS-2后,MTT法测定细胞增殖情况、磷酸对硝基苯酯(PNPP)法检测碱性磷酸酶(ALP)活性,酶联免疫吸附法(ELISA)法检测钙离子(Ca2+)和骨钙素(OTC)的分泌以及用茜素红染色法测定成骨细胞矿化结节数。结果:与空白组比较,酢浆草水提物能明显促进SaOS-2细胞的生长(P<0.01);ALP活性呈时间依赖性增加(P<0.05),以100 mg·L-1剂量组最为显著(P<0.01);增强了细胞分泌Ca2+(P<0.05)和OTC的能力(P<0.01);并且酢浆草中、高剂量组可以明显促进矿化结节形成(P<0.05)。结论:酢浆草水提物在体外可以促进成骨细胞的增殖、分化和矿化,提示酢浆草可能具有促进骨形成的能力。     

补骨脂提取物对体外培养小鼠成骨细胞株MC3T3-E1细胞分化的影响

目的:研究补骨脂提取物对体外培养新生小鼠颅骨Mc313.E1细胞分化的影响.方法:以小鼠成骨细胞株Mc3T3-E1作为药物筛选的细胞模型,将培养的Mc3T3-E1小鼠成骨细胞分为4组:空白组,补骨脂提取物(含生药)0.02,O.2,2 g·L~(-1)不同剂量组.检测细胞ALP,I型胶原蛋白的含量,采用real-time PCR测定补骨脂提取物对MC3r13.E1细胞AIJP,I型胶原蛋白及骨钙素mRNA表达的影响.结果:补骨脂提取物生药剂量O.2 g·L~(-1),能提高MX3T3-E1细胞ALP的活性,促进I型胶原蛋白分泌.同时分别在7～14 d对Mc3r13-E1细胞ALP,以及在14-21 d I型胶原蛋白、骨钙素mRNA表达有明显的促进作用.结论:中药补骨脂提取物能有效促进成骨细胞的分化,为研究中药补骨脂的作用机制提供参考.     

中药Liu-OGN对体外成人成骨细胞增殖和分化的影响

目的：研究不同浓度中药Liu-OGN对体外成人成骨细胞增殖和分化的影响。方法：采用胶原-胰蛋白酶消化法获得成人松质骨中的成骨细胞，并进行纯化和培养，然后使用含不同浓度Liu-OGN药物血清的培养液进行培养，通过倒置相差显微镜和电镜观察细胞生长情况，采用MTT法检测细胞增殖情况，通过碱性磷酸酶和骨钙素等生化指标测定细胞分化情况，结果：获得的成人成骨细胞生长情况良好，生化指标稳定可靠，纯化效果佳，可以满足相关研究的需要，进一步的研究结果表明，在成骨细胞密度为10000/ml的条件下，与不加药对照组比较，10%、20%Liu-OGN中药组和地塞米松组的成骨细胞体积增大，上清液中碱性磷酸酶和骨钙素含量均明显增高；但各Liu-OGN中药组成骨细胞的增殖情况无明显变化；结论：中药Liu-OGN对体外成人成骨细胞的分化有明显地促进作用，可以作为促进骨形成的药物使用。     

牛膝甾酮对小鼠MC3T3-E1细胞成骨分化和增殖的影响

目的研究牛膝甾酮(IS)对小鼠成骨细胞系(MC3T3-E1细胞)增殖、分化的影响及其可能的分子机制。方法将MC3T3-E1细胞分为对照组及IS不同浓度组(10~(-4)～10~(-1)ng/μL),24、48 h后采用细胞计数(CCK8)试剂盒检测细胞增殖,7、14天后采用碱性磷酸酶(ALP)活性试剂盒检测ALP活性,茜素红染色检测矿化结节,原位末端标记(TUNEL)试剂盒检测细胞凋亡情况,实时荧光定量PCR(RT-qPCR)检测成骨分化相关标志物Ⅰ型胶原(CollagenⅠ)、骨保护素(OPG)、骨桥蛋白(OPN)、骨钙素(OCN)、ALP以及雌激素受体alpha(ERα)、雌激素受体beta(ERβ) mRNA的表达水平。结果与对照组比较,在干预48 h后,10~(-3)、10~(-2)、10~(-1)ng/μL IS能抑制细胞的增殖(P0.05),但对细胞凋亡无明显影响。10~(-4)、10~(-3)、10~(-2)、10~(-1)ng/μL IS均能够促进ALP活性及矿化结节的形成(P0.05),亦能促进ERα、OPG、CollagenⅠ、ALP mRNA表达(P0.05);10~(-3)、10~(-2)、10~(-1)ng/μL IS均能促进OPN、OCN mRNA表达(P0.05),10~(-1)ng/μL IS能够促进ERβmRNA表达(P0.05)。结论 IS能促进MC3T3-E1细胞的成骨分化,其可能机制与上调成骨分化标志物及提高ERαmRNA表达有关。     

骨碎补总黄酮含药血清对成骨细胞增殖、分化、周期及调亡的影响

目的:观察含骨碎补总黄酮大鼠血清对体外培养SD大鼠成骨细胞增殖、分化、周期及凋亡的影响.方法:取新生的SD大鼠的颅骨,胶原酶法培养成骨细胞.应用MTT法、对硝基苯磷酸盐法(PNPP)及流式细胞术观察不同浓度的含骨碎补总黄酮大鼠血清在不同时段对体外培养成骨细胞的光密度D(λ)值、碱性磷酸酶(ALP)活性及细胞周期和凋亡的影响.结果:不同浓度的骨碎补总黄酮大鼠含药血清均可促进成骨细胞增殖、分化,使处于DNA合成期细胞比例增加,使早期和晚期凋亡细胞比例减少,与对照组比较均具有统计学意义(P＜0.05). 结论: 骨碎补总黄酮含药血清具有促进体外成骨细胞增殖、分化,抑制成骨细胞凋亡的作用,即具有抗骨质疏松活性.     

骨碎补总黄酮对高糖作用下成骨细胞分化及ERK_(1/2)和p38蛋白激酶表达的影响

目的:研究骨碎补总黄酮(TFDF)对高浓度葡萄糖作用下成骨细胞(OB)分化活性及对细胞外信号调节蛋白激酶(ERK1/2)和p38丝裂原活化蛋白激酶(p38MAPK)信号蛋白的影响,为TFDF能否用来防治糖尿病性骨质疏松提供细胞分子学依据。方法:二次酶消化法分离培养新生SD大鼠颅骨OB及活性观察;MTT法检测TFDF对OB的细胞毒性,确定实验药物在培养基中的添加剂量;pNPP、ELISA、茜素红染色法分别检测不同浓度葡萄糖对OB碱性磷酸酶活性(ALP)、Ⅰ型胶原(ColⅠ)、骨钙素(BGP)以及矿化能力的影响,并观察TFDF对高糖作用下OB分化活性的干预作用;Western-blot检测TFDF对高糖作用下OB ERK1/2和p38蛋白磷酸化的情况。结果:原代分离培养新生SD大鼠OB,具有典型的OB分化特性;根据TFDF对OB的毒性试验,选择TFDF的浓度25、50、100 mg/L为实验梯度浓度;葡萄糖(25、50 mmol/L)可以降低OB表达ALP、ColⅠ、BGP,降低其矿化能力;TFDF能剂量依赖性的提高高糖(25 mmol/L)作用下OB表达ALP、ColⅠ、BGP和矿化能力;TFDF(50 mg/L)能提高高糖(25 mmol/L)作用下p38和ERK1/2的蛋白磷酸化。结论:高糖可以降低OB分化和矿化能力,TFDF可提高高糖作用下OB的分化和矿化能力,其作用机理可能和提高p38和ERK1/2信号蛋白的磷酸化有关。     

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。     

Stimulation of osteoblastic differentiation and mineralization in MC3T3-E1 cells by yeast hydrolysate

In a previous study, it was reported that yeast hydrolysate (YH) was effective in promoting bone growth in Sprague‐Dawley (SD) rats. To further clarify the mechanism of YH, the effects of YH on proliferation, differentiation and gene expression in vitro were investigated using osteoblastic cell lines (MC3T3‐E1). Cell proliferation increased significantly as much as 110% of the basal value when cells were treated with 100 µg/mL of YH. Alkaline phosphatase (ALP) activity increased significantly with a YH concentration of 25–100 µg/mL, and the activity increased 152% that of the control at 100 µg/mL. The calcium content increased as much as 129% at 100 µg/mL YH. The gene expression levels of ALP and collagen type II (COL II) significantly increased approximately 1.3‐fold and 1.7‐fold of control, respectively, at 100 µg/mL. YH increased significantly the mRNA level of bone sialoprotein (BSP) but not in a dose‐dependent manner. The mRNA levels of bone morphogenetic proteins (BMP)‐2, BMP‐4, collagen type I (COL I) and osteonectin (ON) did not increase. In summary, YH increased the proliferation of osteoblasts and directly stimulated ALP and bone matrix proteins (e.g. BSP, COL II), and these increases trigger osteoblastic differentiation (e.g. mineralized nodule formation). Copyright © 2010 John Wiley & Sons, Ltd.     

骨碎补提取液对实验性牙槽骨吸收疗效的研究

目的 :建立实验性大鼠牙槽骨吸收模型 ,了解中药骨碎补 (DFS)对大鼠牙槽骨吸收的疗效。方法 :采用SD大鼠局部注射大肠杆菌内毒素 (E-LPS)建立牙槽骨吸收模型。制备DFS水相提取液。建模大鼠用DFS水提液每天灌胃 1次 ,分别用药 10 ,2 0 ,30d。采用血清碱性磷酸酶 (ALP)活性、钙离子 (Ca²⁺ )和骨钙素 (OC)浓度测定 ,以及牙体-牙周联合标本骨密度 (BMD)、抗酒石酸酸性磷酸酶 (TRAP)染色破骨细胞计数、HE染色组织病理学检查 ,评价DFS水提液治疗实验性牙槽骨吸收的疗效。结果 :与相应阴性对照组比较 ,用药 10d大鼠的实验牙位骨密度显著升高 (P <0 .0 5 ) ,牙周破骨细胞消失 (P <0 .0 5 ) ,Howship陷窝明显减少 ,大多出现根分叉区有成骨细胞附着的新生未钙化骨样基质 ,且第 20 ,30天检查结果与第 10天相似。用药 10～ 30d大鼠的血清标本中ALP活性、Ca²⁺ 和OC浓度与对照组比较均无显著性差异 (P >0 .0 5 )。结论 :DFS水提液对大鼠实验性牙槽骨吸收有明确的疗效 ,能抑制骨质吸收、促进骨质再生。血清ALP活性、Ca²⁺和OC浓度不能作为观察动物模型中牙槽骨吸收及再生的有效指标。     

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.     

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.     

Atractylodes japonica root extract protects osteoblastic MC3T3‐E1 cells against hydrogen peroxide‐induced inhibition of osteoblastic differentiation

The rhizome of Atractylodes japonica Koidz. has been used traditionally for the treatment of water retention in the body and is known to have estrogen‐like activity. In the present study, the protective effects of A. japonica root extract on the response of osteoblasts to oxidative stress were evaluated. Osteoblastic MC3T3‐E1 cells were incubated with hydrogen peroxide and/or A. japonica root extract, and markers of osteoblast function and oxidative damage were examined. A. japonica root extract significantly (p < 0.05) increased cell survival, alkaline phosphatase activity, collagen and calcium deposition and decreased the production of receptor activator of nuclear factor‐kB ligand (RANKL), protein carbonyl and malondialdehyde of osteoblastic MC3T3‐E1 cells in the presence of hydrogen peroxide. These results demonstrate that A. japonica root can protect the cells from oxidative stress‐induced toxicity and may have positive effects on skeletal structure. Copyright © 2009 John Wiley & Sons, Ltd.