玉柏石松中甾体化合物对体外培养成骨细胞活性的影响

为研究玉柏石松中甾体化合物豆甾烷-3-酮-21-羧酸(SA)对体外培养小鼠成骨细胞系MC3T3-E1活性的影响,用Alamar Blue法检测了成骨细胞增殖率,碱性磷酸酶试剂盒检测了细胞中碱性磷酸酶活性,茜素红染色检测了成骨细胞矿化水平,荧光定量PCR检测了成骨细胞骨分化相关基因的表达.结果显示:8μmol/L和16μmol/L的SA处理细胞8 d能抑制成骨细胞碱性磷酸活性;处理细胞16 d能提高骨细胞矿化水平.SA抑制成骨早期分化相关基因(Runx-2和Osterix)的表达,促进骨基质蛋白OPN和骨重建相关转录因子(Jun-D,Fra-1和Fra-2)的表达.故SA具有促进骨折愈合的成骨活性,可能通过促进相关转录因子表达,骨折断面旧骨的吸收和骨基质钙化等方式完成.     

bHGA对体外培养成骨细胞活性的影响

为探讨12b-羟基-去-D-杰氏山竹子素A(bHGA)的体外潜在成骨活性,用bHGA处理MC3T3-E1成骨细胞,研究了其对细胞碱性磷酸酶活性、矿化、骨相关基因表达及早期成骨分化相关基因表达水平的影响.结果表明:bHGA处理细胞后,对Runx2 mRNA表达无显著影响,而对Osx mRNA的表达具有一定抑制作用.bHGA能同时促进骨钙蛋白基因OCN和Ⅰ型胶原Col 1 mRNA的表达,但抑制碱性磷酸酶mRNA的表达,其对骨桥蛋白基因OPN mRNA的表达没有显著影响.说明bHGA不利于骨早期分化,但它通过促进骨钙蛋白和I型胶原mRNA的表达而促进骨成熟.     

绿原酸对体外培养成骨细胞活性的影响

为研究接骨草成分之一绿原酸(CGA)对成骨细胞MC3T3-E1活性的影响,采用MTT法检测11.02,22.05,44.09,88.19μmol/L CGA对成骨细胞增殖率,碱性磷酸酶(ALP)试剂盒检测成骨细胞ALP活性,实时定量PCR检测成骨细胞骨相关基因的表达.结果表明:11.02～88.19μmol/L CGA对成骨细胞增殖具有明显的促进作用.培养2d,44.09μmol/L CGA能促进ALP表达上调;22.05,44.09μmol/L CGA能促进ALP、Runx2、Osterix、c-jun和c-fos基因的表达;培养6 d,44.09μmol/L CGA能促进c-fos基因的表达;在整个培养过程中,I型胶原(Col-I)基因的表达具有浓度和时间依赖性.故CGA具有一定的成骨活性,是接骨草的成骨活性成分之一.     

26-失碳-8-氧代-α-芒柄花萜醇对体外培养成骨细胞活性的影响

为研究玉柏石松提取物26-失碳-8-氧代-α-芒柄花萜醇(26-NO-Ono)对成骨细胞活性的影响,采用MTT检测不同浓度26-NO-Ono(3.33,6.66,13.32,26.64μmol/L)对成骨细胞增殖率,碱性磷酸酶(ALP)试剂盒检测成骨细胞内ALP活性,荧光定量PCR检测成骨细胞骨相关基因表达.结果表明:26-NO-Ono给药1d可促进成骨细胞增殖,给药3d可促进成骨细胞ALP活性.26-NO-Ono处理3d和9d会抑制骨涎蛋白(BSP)、I型胶原蛋白(Col-I)以及骨钙素蛋白(OCN)的基因表达;处理6d会促进上述基因的表达.26-NO-Ono长期处理(6d和9d)可以抑制骨桥蛋白(OPN)的基因表达,说明26-NO-Ono对成骨细胞的成骨活性的影响呈时间依赖性,剂量依赖性和细胞分化状态依赖性.     

山芝烯二醇对体外培养成骨细胞成骨活性的影响

为研究玉柏石松提取物山芝烯二醇对体外培养小鼠颅骨成骨细胞活性的影响,采用MTT法、碱性磷酸酶（ALP）试剂盒、荧光定量PCR检测不同浓度药物作用不同时间后成骨细胞增殖、ALP活性和成骨活性相关基因,如原癌基因（c-jun、c-fos）、成骨转录因子（Osterix）、碱性磷酸酶（ALP）、Ⅰ型胶原（Col-Ⅰ）、骨钙素（OC）表达.结果显示：山芝烯二醇处理早期（3 d）促进成骨细胞增殖,促进c-jun、c-fos基因表达,先抑制后促进Osterix基因表达;晚期（9 d）促进ALP活性和ALP、Col-Ⅰ基因表达,对OC基因无明显影响.说明山芝烯二醇可以促进成骨细胞的增值,ALP活性及部分骨相关基因表达,是玉柏石松促进骨愈合的有效成分之一.     

利用报告基因表达系统快速鉴定骨诱导活性材料

通过构建真核表达质粒,使增强型绿色荧光蛋白(Enhanced Green Fluorescent Protein, EGFP)报告基因的表达受骨形成相关转录因子Cbfa1基因启动子的调控,再用该质粒转染大鼠成肌细胞L6,建立稳定细胞株.在成骨培养基中诱导培养后,该细胞株呈现较明显的绿色荧光,表明细胞在经诱导的成骨分化过程中,Cbfa1基因表达增强,Cbfa1启动子驱动了EGFP的表达.将该细胞株接种到有较强骨诱导活性的羟基磷灰石/磷酸三钙(hydroxyapatitetricalcium phospha     

La^3＋通过重组细胞骨架影响大鼠成骨细胞增殖、分化

研究了稀土离子La^3＋对体外培养的成骨细胞增殖、分化及细胞骨架的影响,并初步探讨了相关机理．用细胞计数法检测了成骨细胞的增殖．用RT-PCR技术测定了碱性磷酸酶（ALP）、骨钙素（OC）、骨桥蛋白（OPN）,骨涎蛋白（BSP）以及cbfa-1mRNA水平．采用激光扫描共聚焦显微镜观察了细胞中F肌动蛋白（F-actin）的变化．结果显示：La^3＋在48h促进了成骨细胞增殖;在4d促进了早期分化指标ALP,BSP和拍肠-1mRNA的表达;在21d促进了晚期分化指标OC和OPNmRNA的表达．与此同时,La^3＋使成骨细胞骨架发生重组．另外,Western Blot分析证实La^3＋作用于成骨细胞短时间即可激活粘着斑激酶（FAK）酪氨酸磷酸化．结果提示,La^3＋通过提高FAK酪氨酸的磷酸化水平,改变细胞骨架的分布和聚合,从而促进成骨细胞的增殖和分化．     

人间充质干细胞成骨分化早期HOX家族基因转录的表观遗传调控

人间充质干细胞(human mesenchymal stem cells,hMSCs)是一类具有多分化潜能的成体干细胞,在体内外可以被人工定向诱导分化成多种不同的细胞.有报道表明,在干细胞分化的过程中,细胞核内染色质发生重塑.HOX家族基因作为一类转录因子,在胚胎发育以及细胞分化过程中发挥着十分重要作用.通过体外定向诱导骨髓间充质干细胞向成骨细胞分化,对比分化前后细胞中HOX家族基因的表达状况,发现HOX家族基因的表达水平在hMSCs早期成骨分化过程中显著下降.进一步的研究发现,HOX家族基因的这种表达变化是由其启动子区的组蛋白H3-Lys9乙酰化和二甲基化水平发生变化而导致的.一系列实验证据表明,在间充质干细胞的成骨分化过程中,HOX家族基因表达受到抑制,而这种抑制作用是与其分化过程中发生的染色质重塑事件密切相关的.     

稀土离子对体外培养的成骨细胞增殖、分化和功能表达的影响

采用噻唑蓝(MTT)法、碱性磷酸酯酶比活性的定量测定、细胞周期测定、矿化结节计数、扫描和透射电子显微镜观察等方法研究了不同浓度稀土离子(Ln3 ,包括La3 ,Sm3 ,Dy3 ,Nd3 ,Er3 和Gd3 )对成骨细胞UMR 106增殖、分化和功能表达的影响.结果表明,浓度在1.00×10-4 mol/L时,各稀土离子均表现出抑制成骨细胞增殖的作用. 当浓度降低到1.00×10-9～1.00×10-5 mol/L范围时,则表现出促进作用. 稀土离子(Ln3 )浓度为1.00×10-7及1.00×10-5 mol/L时,显著增强碱性磷酸酯酶比活性(P<0.01). 对其中一个稀土离子La3 作了进一步机理研究,发现极低浓度下(1.00×10-8及1.00×10-9 mol/L)的La3 促进成骨细胞从G0/G1期向S期过渡,并且增强成骨细胞的矿化能力(P<0.001). 扫描电子显微镜和透射电子显微镜结果表明,高浓度(1.00×10-4 mol/L)La3 可使成骨细胞损伤,可见表面变为光滑,微绒毛消失, 细胞膜不完整, 内质网扩张,核膜外凸,核周隙扩张. 低浓度(1.00×10-9 mol/L) La3 对成骨细胞无明显影响. 所得结果提示稀土离子(Ln3 )能够促进体外培养的成骨细胞的增殖、分化和功能表达,呈现出浓度依赖的两面性,并与稀土物种有关.     

不同应变水平拉伸对成骨细胞生理功能的影响

采用四点弯曲加载装置对大鼠颅盖骨中分离出的成骨细胞施加周期性拉伸刺激，研究成骨细胞对不同应变水平的拉伸刺激的生理响应。结果表明，500微应变的周期性拉伸刺激促进了成骨细胞的增殖，细胞的^3H－脯氨酸掺入量增加，碱性磷酸酶（ALP）活性和向胞外分泌钙均升高；而1000微应变的周期性拉伸抑制了成骨细胞的增殖、^3H－脯氨酸掺入量、碱性磷酸酶活力和向胞外分泌钙都降低，说明成骨细胞能够分辨不同变水平的力学刺激，500微应变的周期性拉伸刺激有利于细胞的生长和分化，而1000微应变的周期性拉伸对细胞的生理活性有抑制作用。而且细胞在增殖、基质合成和分化、矿化上表现出一致的变化趋势。     

Effects of genistein, daidzein and glycitein on the osteogenic and adipogenic differentiation of bone marrow stromal cells and on the adipogenic trans-differentiation of osteoblasts

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), alkaline phosphatase (ALP) activity and oil red O assays were used to examine the effects of genistein, daidzein and glycitein on the osteogenic and adipogenic differentiation of primary mouse bone marrow stromal cells (MSCs) and the adipogenic trans-differentiation of primary mouse osteoblasts. The results indicated that daidzein, genistein and glycitein at concentrations from 1×10^-8 mol/L to 1×10^-5 mol/L promoted the proliferation of MSCs and osteoblasts; genistein, daidzein and glycitein promoted osteogenic differentiation and inhibited adipogenic differentiation of MSCs, and inhibited adipocytic transdifferentiation of osteoblasts at appropriate concentrations as 17β-estradiol. It suggests that genistein, daidzein and glycitein regulate a dual differentiational process of MSCs into the osteogenic and adipogenic lineages, and trans-differentiational process of primary osteoblasts into the adipocyte lineages, causing a lineage shift toward osteoblast. Protective effects of them on bone may be mediated by a reversal of adipogenesis which may promote the proliferation, differentiation and mineralization of osteoblasts, and make adipocytes secrete less cytokines which may promote osteoclast formation and activation. In addition, the results also indicated that genistein, daidzein and glycitein may be helpful in preventing the development of steroid induced osteonecrosis.     

Osteogenic differentiation of mesenchymal stem cells promoted by overexpression of connective tissue growth factor

Objective: Large segmental bone defect repair remains a clinical and scientific challenge with increasing interest focusing on combining gene transfection with tissue engineering techniques. The aim of this study is to investigate the effect of connective tissue growth factor (CTGF) on the proliferation and osteogenic differentiation of the bone marrow mesenchymal stem cells (MSCs). Methods: A CTGF-expressing plasmid (pCTGF) was constructed and transfected into MSCs. Then expressions of bone morphogenesis-related genes, proliferation rate, alkaline phosphatase activity, and mineralization were examined to evaluate the osteogenic potential of the CTGF gene-modified MSCs. Results: Overexpression of CTGF was confirmed in pCTGF-MSCs. pCTGF transfection significantly enhanced the proliferation rates of pCTGF-MSCs (P<0.05). CTGF induced a 7.5-fold increase in cell migration over control (P<0.05). pCTGF transfection enhanced the expression of bone matrix proteins, such as bone sialoprotein, osteocalcin, and collagen type I in MSCs. The levels of alkaline phosphatase (ALP) activities of pCTGF-MSCs at the 1st and 2nd weeks were 4.0- and 3.0-fold higher than those of MSCs cultured in OS-medium, significantly higher than those of mock-MSCs and normal control MSCs (P<0.05). Overexpression of CTGF in MSCs enhanced the capability to form mineralized nodules. Conclusion: Overexpression of CTGF could improve the osteogenic differentiation ability of MSCs, and the CTGF gene-modified MSCs are potential as novel cell resources of bone tissue engineering.     

不同诱导条件对树鼩骨髓间充质干细胞向成骨细胞分化的影响

目的 探讨不同诱导条件对树鼩骨髓间充质干细胞（BM-MSCs）体外向成骨细胞分化的影响。方法 取 P3代树鼩骨髓间充质干细胞分成7组进行诱导。A组：高糖DMEM+1μmol/L地塞米松+100μmol/L维生素C+10 mmol/Lβ-磷酸甘油钠;B组：高糖 DMEM+50 ng/mL BMP-2;C组：高糖 DMEM+80 ng/mL BMP-2;D组：高糖DMEM+50μmol/L维生素C+10 mmol/Lβ-磷酸甘油钠+20ng/m L BMP-2;E组：高糖 DMEM+1μmol/L地塞米松 +100μmol/L维生素C+20 mmol/Lβ-磷酸甘油钠;F组：高糖DMEM+100μmol/L地塞米松+100μmol/L维生素 C+10 mmol/Lβ-磷酸甘油钠;G组：DMEM/F12+0.1μmol/L地塞米松 +50μmol/L维生素C+10 mmol/Lβ-磷酸甘油钠。诱导18 d后进行碱性磷酸酶和茜素红染色鉴定其诱导分化情况。结果 每一组碱性磷酸酶染色呈不同程度的阳性,茜素红染色可在A、B、C、D和E组观察到明显矿化结节。结论 A组、B组、C组、D组和 E组可以诱导树鼩BM-MSCs向成骨细胞分化,其中以A组和B组效果最为突出。     

Effect of Dy3+on osteogenic and adipogenic differentiation of mouse primary bone marrow stromal cells and 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, mineralized function, Oil Red O stain and measurement were employed to assess the effect of Dy³⁺ on the osteogenic and adipogenic differentiation of mouse primary bone marrow stromal cells (BMSCs) and the adipogenic trans-differentiation of mouse primary osteoblasts (OBs). The results showed that Dy³⁺ had no effect on BMSC proliferation at concentrations of 1×10⁻⁸ and 1×10⁻⁵ mol/L, but inhibited BMSC proliferation at other concentrations. Dy³⁺ had no effect on OB proliferation at concentrations of 1×10⁻¹⁰ and 1×10⁻⁹ mol/L, but inhibited OB proliferation at other concentrations. Dy³⁺ had no effect on the osteogenic differentiation of BMSCs at concentrations of 1×10⁻⁹ and 1×10⁻⁷ mol/L, and promoted osteogenic differentiation of BMSCs at other concentrations at the 7th day. The osteogenic differentiation of BMSCs was inhibited by Dy³⁺ at concentration of 1×10⁻⁵ mol/L at the 14th day, but promoted osteogenic differentiation of BMSCs at concentrations of 1×10⁻⁹, 1×10⁻⁸, 1×10⁻⁷ and 1×10⁻⁶ mol/L with the maximal effect at concentration of 10⁻⁶ mol/L. Dy³⁺ promoted mineralized function of BMSCs at any concentration. Dy³⁺ had no effect on adipogenic differentiation of BMSCs at concentration of 1×10⁻⁷ mol/L, but inhibited adipogenic differentiation of BMSCs at other concentrations. Dy³⁺ inhibited adipocytic trans-differentiation of OBs at any concentration, suggesting that Dy³⁺ had protective effect on bone and the protective effect on bone may be mediated by modulating differentiation of BMSCs away from the adipocyte and inhibiting adipocytic trans-differentiation of OBs which may promote differentiation and mineralization of OBs. These results may be valuable for better understanding the mechanism of the effect of Dy³⁺ on pathogenesis of osteoporosis. Supported by the Foundation for Key Program of Ministry of Education of China (Grant No. 208018)     

Notch signaling stimulates osteogenic differentiation of human bone marrow-derived mesenchymal stem cells

Notch signaling is one of the most important pathways mediating cell determination and differentiation.In this study, the roles of Notch signal in the regulation of osteogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs) were investigated. The expression of Notch1, Jaggedl and DTXI detected by reverse transcrip-tion polymerase chain reaction (RT-PCR) suggested that Notch signal might exhibit a physiological regulatory role in the differentiation of MSCs. Constitutive expression of the intracellular domain of Notchl (ICN), the active form of Notchl protein, can activate Notch signal in cells without ligands‘ binding, hMSCs were isolated, expanded, and infected with retrovirus carrying green fluorescent protein (GFP) gene or ICN. Overexpression of ICN in hMSCs resulted in enhanced osteogenic differentiation induced by dexamethasone (Dex), which was characterized by an increase of cellular alkaline phosphatase (ALP) activity and calcium deposition. These results indicate that Notch stimulates differentiation of MSCs into osteoblasts.     

Synergistic effect of trace elements and flavonoids from Epimedium koreanum Nakai on primary osteoblasts

Several trace elements, particularly, manganese （Mn） and zinc （Zn）, are essential in bone metabolism as cofactors for specific enzymes. It has been reported that there exists the relationship between osteoporosis and trace element-deficiency and the efficacy of Ca, Mn and Zn supplementation on spinal bone mineral density in postmenopausal women. Traditional Chinese medicines （TCM）, such as Herba epimedii, were proved to be effective for prevention of osteoprosis in vivo; however, the efficacy of the main constituents and/or crude extract was not ideal in vitro, which suggested that they may work in another way. The purpose of the present study was to examine whether the combination of icariin and total flavonoids （TF） from Herba epimedii with mineral elements, which were abundant in Herba epimedii, would have a more beneficial effect on the viability and differentiation of primary osteoblasts than either agent alone, and to analyze the dada for a possible synergistic, additive or antagonistic effect. The combinations of 10 μmol/L Zn, Ca and Mn with icariin and total flavonoids greatly improved the cell viability and meanwhile dramatically enhanced the alkaline phosphatase activity as compared to each agent alone. On the other hand, an increased cell growth inhibition was also observed by combining 0.1 μmol/L, 1 pmol/L Zn with 10μmol/L icariin, and 10 μmol/L Mn with 0.06 μg/mL total flavonoids. Meanwhile a decreased alkaline phosphatase activity was also found in several icariin-Zn/Mn and total flavonoids-Zn/Ca/Mn combinations. These results suggested that mineral elements （Zn, Ca, Mn） greatly enhanced the efficacy of icariin and total flavonoids from Herba epimedii on the viability and differentiation of primary osteoblasts by certain combinations.