甲氧补骨脂素对大鼠成骨细胞生物活性影响的实验研究

为探讨甲氧补骨脂素对体外培养新生大鼠颅骨成骨细胞增殖与分化作用的影响,用改良的组织块培养法分离培养新生大鼠颅骨成骨细胞,在成骨细胞体系中以不同浓度加入甲氧补骨脂素,MTT法检测加药后不同时间细胞的增殖情况;用对硝基苯二钠基质动力学法(PNPP)测定细胞内碱性磷酸酶的活性,用改良的Lowry法测蛋白含量;用放射免疫法测定细胞内骨钙素含量。结果显示:与对照组相比,甲氧补骨脂素组在24 h和36 h时促进体外大鼠成骨细胞增殖的作用更明显;在24、48 h和72 h时均能提高成骨细胞碱性磷酸酶活性(ALP)和骨钙素(BGP)的分泌。甲氧补骨脂素对体外培养的大鼠成骨细胞的增殖与分化均有明显的促进作用。     

异补骨脂素加锌对大鼠成骨细胞增殖与分化的影响

为探讨异补骨脂素加锌对体外培养新生大鼠颅骨成骨细胞增殖与分化作用的影响,用改良的组织块培养法分离培养新生大鼠颅骨成骨细胞,在成骨细胞体系中以不同浓度加入异补骨脂素与锌,MTT法检测加药后不同时间成骨细胞的增殖情况;用对硝基苯二钠基质动力学法(PNPP)测定细胞内碱性磷酸酶的活性,用改良的Lowry法测蛋白含量.结果显示:异补骨脂素加锌较单纯应用异补骨脂素或硫酸锌在24和48 h时促体外大鼠成骨细胞增殖的作用更加明显;在48和72 h时能促进成骨细胞碱性磷酸酶活性(ALP),其中ALP的测定在72h的活性更为显著.与单纯应用异补骨脂素或者锌相比,异补骨脂素与锌联合应用能够协同增效,对体外培养的成骨细胞的增殖与分化作用更加显著.     

补骨脂素对大鼠成骨细胞增殖与分化的影响

为探讨补骨脂素体外对大鼠成骨细胞增殖与分化的影响,用改良的组织块法分离培养新生大鼠颅骨成骨细胞,补骨脂素以不同浓度加入细胞培养体系,作用不同时间后,用MTT法检测成骨细胞的增殖情况;用对硝基苯二钠基质动力学法测定细胞内碱性磷酸酶的活性,用改良的Lowry法测蛋白含量。补骨脂素浓度在1μmol/L 24 h,5~20μmol/L浓度范围内48 h,1、10、20μmol/L浓度范围内72 h促进成骨细胞增殖,在10~15μmol/L范围内48 h及72 h提高成骨细胞内碱性磷酸酶的活性。补骨脂素体外能促进成骨细胞的增殖与分化。     

大豆甙元对大鼠成骨细胞增殖与分化的影响

目的探讨大豆甙元体外对大鼠成骨细胞增殖与分化的影响。方法用改良的组织块法分离培养新生大鼠颅骨成骨细胞,大豆甙元以不同浓度加入细胞培养体系,作用不同时间后,用MTT法检测成骨细胞的增殖情况;用放射免疫法(RIA)测定细胞外骨钙素(BGP)的含量,用改良的Lowry法测蛋白含量。结果大豆甙元1×10(-5)～1×10(-9)mol／L浓度范围内48h,72h促进成骨细胞增殖,在1×10(-7)～1×10(-9)mol／L范围内48h和72h提高成骨细胞外骨钙素含量。结论大豆甙元体外能促进成骨细胞的增殖与分化。     

五味子乙素对大鼠成骨细胞增殖分化的影响

目的:探讨五味子乙素体外对大鼠成骨细胞增殖与分化的影响。方法:用改良的组织块法分离培养新生大鼠颅骨成骨细胞,五味子乙素以不同浓度加入细胞培养体系,作用不同时间后,用MTT法检测成骨细胞的增殖情况;用对硝基苯二钠基质动力学法(PNPP)测定细胞内碱性磷酸酶的活性,用改良的Lowry法测蛋白含量。结果:五味子乙素在0.75×10-4 mol/L 24 h,48 h及72 h,及0.75×10-5mol/L 24 h促进成骨细胞的增殖,在0.75×10-6mol/L24 h提高成骨细胞内碱性磷酸酶的活性。结论:五味子乙素体外能促进成骨细胞的增殖与分化。     

五味子甲素对大鼠成骨细胞增殖分化的影响

目的　探讨五味子甲素体外对大鼠成骨细胞增殖与分化的影响。方法　用改良的组织块法分离培养新生大鼠颅骨成骨细胞 ,五味子甲素以不同浓度加入细胞培养体系 ,作用不同时间后 ,用MTT法检测成骨细胞的增殖情况 ;用对硝基苯二钠基质动力学法 (PNPP)测定细胞内碱性磷酸酶的活性 ,用改良的Lowry法测蛋白含量。 结果　五味子甲素在 0 75× 10 -5mol/L及 0 75× 10 -9mol/L浓度范围内 2 4h ,72h ,0 75× 10 -4 ～ 0 75× 10 -7mol/L 48h促进成骨细胞增殖 ,在 0 75× 10 -5～ 0 75× 10 -6mol/L浓度范围内 48h提高成骨细胞内碱性磷酸酶的活性。结论　五味子甲素体外能促进成骨细胞的增殖与分化。     

山萘酚对大鼠成骨细胞增殖与分化的影响

为探讨山萘酚体外对大鼠成骨细胞增殖与分化的影响及中药菟丝子补肾壮骨作用的物质基础,用改良的组织块法分离培养新生大鼠颅骨成骨细胞,山萘酚以不同浓度加入细胞培养体系,作用不同时问后,用MTF法检测成骨细胞的增殖情况;用对硝基苯二钠基质动力学法测定细胞内碱性磷酸酶的活性,用改良的Lowry法测蛋白含量。山萘酚在浓度1×10^-5mol／L24h,1×10^-5～1×10^-7mol／L浓度范围内48h促进成骨细胞增殖,在浓度1×10^-9moL／L48h及72h提高成骨细胞内碱性磷酸酶的活性。山萘酚体外能促进大鼠成骨细胞的增殖与分化,山萘酚可能是菟丝子补肾壮骨作用的活性成份之一。     

佛手柑内酯对大鼠成骨细胞增殖与分化的影响

为研究佛手柑内酯对大鼠成骨细胞增殖、分化的影响。原代培养新生大鼠颅骨细胞,利用MTT法、微量酶标法、酶联免疫吸附法(ELISA)、q PCR等方法分别测定不同浓度佛手柑内酯对大鼠成骨细胞增殖,碱性磷酸酶(ALP),骨钙素(BGP),Ⅰ型胶原mRNA(CollagenⅠmRNA)表达的影响。结果显示,与对照组相比,佛手柑内酯作用于细胞24、48、72 h均对成骨细胞的增殖有促进作用(P0.05);作用48、72 h均能促进ALP、BGP、CollagenⅠmRNA表达(P0.05)。表明佛手柑内酯可促进大鼠成骨细胞的增殖和分化,为防治骨质疏松症的新药研究提供理论依据。     

异补骨脂素加锌对大鼠成骨细胞相关细胞因子表达影响的实验研究

为探讨异补骨脂素加锌对体外培养新生大鼠颅骨成骨细胞相关基因表达的影响,用改良的组织块培养法分离培养新生大鼠颅骨成骨细胞,在成骨细胞体系中加入异补骨脂素与锌,以雌激素为阳性对照,空白组为阴性对照。结果显示:异补骨脂素加锌较单纯应用异补骨脂素或硫酸锌在48 h时促体外大鼠成骨细胞Ⅰ型胶原的表达;异补骨脂素加锌组可以明显上调大鼠成骨细胞TGF-β1的细胞信号转导因子Smad4 mRNA的表达(P0.01);能促进Runx2/Cbfa1 mRNA的表达(P0.05)。与空白对照组相比,异补骨脂素组,异补骨脂素加锌组均能增强Osterix mRNA的表达(P0.05)。与单纯应用异补骨脂素或者锌相比,异补骨脂素与锌联合应用能够协同增效,对促进体外培养的成骨细胞相关转录因子的表达更加明显,探讨了异补骨脂素及其与锌配伍调节骨代谢的分子机制,为提高骨质疏松症的临床疗效以及抗骨质疏松新药的开发提供了实验依据。     

丹皮酚对过氧亚硝基阴离子致大鼠成骨细胞分化抑制的影响

探讨丹皮酚（Pae）拮抗过氧亚硝基阴离子（ONOO^-）对体外培养大鼠成骨细胞分化的影响。用改良的组织块法分离培养新生大鼠颅骨成骨细胞,采用淬灭流动反应方法体外制备ONOO^-,以不同终浓度加入成骨细胞培养体系,在作用不同时间后,用对硝基苯二钠动力学（PNPP）法检测细胞内碱性磷酸酶（ALP）的活性,用Lowry法测定蛋白含量,并以不同终浓度Pae消除ONOO^-（1000μmoL/L）对成骨细胞分化的影响。结果显示,不同浓度的ONOO^-（50-1000μmoL/L）均能抑制碱性磷酸酶的活性,影响分化;高浓度的丹皮酚（10^-3-10^-6mol/L）能消除ONOO^-（1000μmoL/L）对碱性磷酸酶活性的抑制,拮抗ONOO^-抑制成骨细胞分化的作用。     

锌对铅染毒新生大鼠成骨细胞增殖分化的影响

目的:探讨铅锌联合染毒对乳鼠颅骨成骨细胞增殖分化的影响。方法:分离并培养原代成骨细胞,加入不同浓度铅、锌培养48h,检测其对成骨细胞增殖的作用;用碱性磷酸酶试剂盒检测ALP活力。结果:在染铅48h后,当铅浓度≥10μmol/L时,细胞增殖功能下降(P<0.05);加锌干预48h后,铅+锌组细胞增殖功能均高于各自单独染铅组,其中铅(1μmol/L、10μmol/L)+锌(50μmol/L)组、铅(10)+锌(100)组与对照组间的差异具有统计学意义(P<0.05)。铅干预48h后,100μmol/L铅组的ALP活力显著下(P<0.05),给予锌干预的铅锌联合染毒组,各组ALP活力均有增加,其中铅(1μmol/L、10μmol/L)+锌(50μmol/L)组ALP活力均高于对照组,而铅(100μmol/L)+锌(50μmol/L)组ALP活力低于对照组,差异均有统计学意义(P<0.05)。结论:铅对成骨细胞有毒性作用,影响其增殖和分化功能;50μmol/L锌在一定程度上可以拮抗铅对成骨细胞增殖和分化功能的损伤,且对ALP活力的作用更显著,为铅中毒骨病的防治提供一定的科学依据。     

补骨脂素对TCP磨损颗粒所致大鼠成骨细胞损伤的干预作用及其机制

目的: 探讨补骨脂素(psoralen)对TCP磨损颗粒诱导成骨细胞损伤的影响及其分子机制。方法: 通过消化法从SD大鼠乳鼠颅骨中获取原代的成骨细胞,应用碱性磷酸酶(ALP)染色鉴定成骨细胞。TCP磨损颗粒(0.1 mg/ml)与成骨细胞共孵育48 h构建成骨细胞损伤的体外实验模型,实验随机分为正常对照组(Control)、模型(TCP)组和psoralen(10^-7 mol/L、10^-6 mol/L和10^-5 mol/L)组。WST法和流式细胞术分别检测各组成骨细胞活性变化和凋亡情况;化学比色法检测成骨细胞中ALP活性;各组成骨细胞培养14 d后应用茜素红S染色观察矿化结节形成。Western blot法检测各组成骨细胞中葡萄糖调节蛋白78/94(GRP78/94)、肌醇依赖酶1α(IRE1α)、剪切型X盒结合蛋白1(XBP1s)和磷酸化c-Jun氨基末端激酶(p-JNK)等蛋白质的表达。结果: 与Control组比较,TCP组成骨细胞活性、ALP活性和矿化结节的生成显著降低(P＜0.05),细胞凋亡率、GRP78/94、IRE1α、XBP1s和p-JNK等蛋白质表达明显增加(P＜0.05);与TCP组比较,补骨脂素各组成骨细胞损伤情况明显减轻,细胞凋亡率显著减少(P＜0.05),GRP78/94、IRE1α、XBP1s和p-JNK等蛋白质表达也明显下降(P＜0.05)。结论: 补骨脂素可抑制TCP磨损颗粒诱导的IRE1α-XBP1s-JNK信号通路的激活,阻止TCP颗粒所致的成骨细胞损伤及凋亡。     

成骨细胞对梯度拉伸应变的响应

采用四点弯曲加载装置对原代的大鼠颅盖骨细胞施加周期性的拉伸刺激,并设计了应变呈梯度增加的加载方式,使成骨细胞受到的拉伸应变为500－1500με,每隔2h增加500με,以考察成骨细胞对变化的力学环境的响应。结果表明,在500με下拉伸2－6h促进了成骨细胞的增殖、碱性磷酸酶活力增强和胞外钙基质沉积。对细胞施加应变呈梯度增加的拉伸刺激,则发现当应变从有利于细胞的生长分化水平（500με）变化为不利于细胞生长分化的水平（1000με,1500με）后,细胞的增殖指数、碱性磷酸酶活力和胞外钙基质分泌量都迅速降低,以适应新的力学环境。说明成骨细胞能够分辨不同的应变水平,并相应地调节自身的生理功能,从而表现出对变化的力学环境的适应。     

Effects of different extremely low-frequency electromagnetic fields on osteoblasts

It is well known that the extremely low-frequency electromagnetic field (EMF) can promote the healing of bone fractures, but its mechanism remains poorly understood. The purpose of this study was to examine the response of neonatal rat calvarial bone cells to the rectangular electromagnetic field (REMF), triangular electromagnetic field (TEMF), sinusoidal electromagnetic field (SEMF), and pulsed electromagnetic field (PEMF). The stimulatory effects of EMF were evaluated by the proliferation (methyltetrazolium colorimetric assay), differentiation (alkaline phosphatase (ALP) activity), and mineralization (area of mineralized nodules of the cells). REMF treatment of osteoblasts increased cellular proliferation and decreased ALP activity (p < 0.05). TEMF had an accelerative effect on the cellular mineralized nodules (p < 0.05). SEMF treatment of osteoblasts decreased the cellular proliferation, increased ALP activity, and suppressed mineralized nodules formation (p < 0.05). PEMF promoted the proliferation of osteoblasts, inhibited their differentiation, and increased the mineralized nodules formation (p < 0.05). Moreover, the effects of PEMF on osteoblasts were concerned with the extracellular calcium, P2 receptor on the membrane, and PLC pathway, but the response of osteoblasts on SEMF was only related to PLC pathway. The results suggested that the waveforms of EMF were the crucial parameters to induce the response of osteoblasts.     

Effect of zinc ion on the osteogenic and adipogenic differentiation of mouse primary bone marrow stromal cells and the adipocytic trans-differentiation of mouse primary osteoblasts

A series of experimental methods including 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test, alkaline phosphatase (ALP) activity measurement and Oil Red O stain and measurement were employed to assess the effect of zinc ion on the osteogenic and adipogenic differentiation of mouse primary bone marrow stromal cells (MSCs) and the adipogenic trans-differentiation of mouse primary osteoblasts. The results showed that except for individual concentrations of zinc ion there was no effect on the proliferation of MSCs and osteoblasts. Zinc ion inhibited the osteogenic differentiation of MSCs at all the concentrations tested. It also inhibited adipogenic differentiation at all concentrations tested except 10(-9)mol/L. Both of the inhibition effects were attenuated with time increasing. Zinc ion depressed adipocytic trans-differentiation of osteoblasts at concentrations of 10(-11) and 10(-10)mol/L, but the effect could be reversed to promote or even be removed when concentration was increased. It suggests that the influence of zinc ion on osteogenic, adipogenic differentiation of MSCs and adipocytic trans-differentiation of osteoblasts depends on zinc ion concentrations and incubation time. The protective effects of zinc ion on bone may be mediated by modulating differentiation of MSCs away from the adipocytes and inhibiting adipocytic trans-differentiation of osteoblasts. This may in turn promote osteoblast formation and reduce secretion of cytokines which may inhibit osteoclast formation and activation. These findings may be valuable for better understanding the mechanism of the effect of zinc ion on bone.     

染料木黄酮对成骨细胞增殖分化的影响

目的：研究染料木黄酮对体外培养乳鼠颅盖骨成骨细胞增殖分化的影响。方法：取乳鼠颅盖骨,采用胶原-胰蛋白酶消化法,进行颅骨成骨细胞培养,取第二代成骨细胞,添加10^-5～10^-7mol／L染料木黄酮,在CO2孵箱中培养48h和72h后MTT比色法测定细胞增殖,培养72h采用^3H-TdR和^H-Pro掺入实验测定DNA和胶原合成。用试剂盒检测细胞裂解液碱性磷酸酶(ALP)活性。结果：染料木黄酮明显增加成骨细胞MTT的吸光度值、^3H-TdR和^3H-Pro的掺入,增加成骨细胞碱性磷酸酶活性。结论：染料木黄酮促进体外培养的乳鼠颅盖骨成骨细胞DNA和胶原的合成,促进增殖和分化。     

Effects of total flavonoids and flavonol glycosides from Epimedium koreanum Nakai on the proliferation and differentiation of primary osteoblasts.

In a bioassay-guided drug screening for anti-osteoporosis activity, eight flavonol glycosides were isolated from Epimedium koreanum Nakai, which is traditionally widely used in China for the treatment of impotence and osteoporosis. The effects of total flavonoids and flavonol glycosides on the proliferation and differentiation of rat calvarial osteoblast-like cells were evaluated by the MTT method and measuring the activity of alkaline phosphatase (ALP activity). Total flavonoids (1.2 x10(-2) to 6.0 x10(-7) mg/ml) and flavonol glycosides (2.0 x10(-5) to 1.0 x10(-9) mol/l) exhibited a strong inhibition on the proliferation of primary osteoblasts at most concentrations. However, the total flavonoids and icariin significantly promoted the differentiation of primary osteoblasts. The results suggested that flavonoids from E. koreanum Nakai may improve the development of osteoblasts by promoting the ALP activity; and icariin might be one of the active constituents facilitating the differentiation of osteoblasts.     

In vitro effects of low frequency electromagnetic fields on osteoblast proliferation and maturation in an inflammatory environment

An in vitro model was set up to investigate the effects of low frequency pulsed electromagnetic fields (PEMF) and its induced electric fields on osteoblast cells under inflammatory conditions. Osteoblasts (7F2) were seeded on top of chitosan scaffolds and co-cultured with macrophage cells (RAW 264.7) growing on the bottom of culture wells, stimulated by lipopolysaccharide to release reactive oxygen species including nitric oxide (NO). The co-culture was exposed to PEMF (magnitude of the magnetic field = 1.5 mT; induced electric voltage = 2.5 mV; frequency = 75 Hz; pulse duration = 1.3 ms) for 9 h. The osteoblasts were examined for their proliferation, viability, alkaline phosphatase (ALP) activity, and genetic expressions of type I collagen (COL I) and osteocalcin (OC), immediately and 7 days after PEMF exposure (days 0 and 7). Macrophage cell viability and NO concentration in the medium were monitored before and after PEMF exposure. The PEMF-exposed co-culture released a significantly higher amount of NO (65 μM) compared to control (17 μM) on day 7. Despite the high level of NO in the medium that was reported to be cytotoxic, PEMF-exposed osteoblasts had enhanced cell proliferation (23%), viability (36%), and COL I mRNA expression (3.4-fold) compared to the controls. The osteoblasts subjected to the PEMF had 41% less ALP activity than the control, which was associated with the active cell proliferation and COL I expression. The expression of OC mRNA was not seen in either the PEMF or control group, indicating cells had not entered the mineralization stage by day 7.     

磷酸三钙涂层镁合金材料的细胞相容性研究

目的:制备磷酸三钙(β-TCP)涂层镁合金材料,评价材料表面的特性及体外的细胞生物适应性。方法 电化学法制备β-TCP涂层镁合金材料(β-TCP-Mg-AI-Zn),观测金属材料表面微观结构特性和能谱分析,小鼠颅骨源成骨细胞与材料直接接触培养,荧光染色观察材料表面细胞生长状况,检测成骨细胞增殖和碱性磷酸酶(ALP)活性。结果 β-TCP涂层Mg-AI-Zn材料表面呈多孔状,材料表面含有镁、钙和磷等元素;成骨细胞与材料直接接触培养24 h及48 h后,材料表面有大量的成骨细胞粘附、伸展、汇合;与Mg-AI-Zn材料比较,β-TCP-Mg-AI-Zn材料明显地促进细胞增殖、显著地增加成骨细胞中ALP活性 (P＜0.05)。结论 β-TCP涂层改善了Mg-AI-Zn镁合金材料表面特性及体外的细胞相容性,有望成为新一代可降解医用金属材料。