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.     

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.     

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.     

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.     

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．     

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。     

La3+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La3 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-1 mRNA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:La3 在48h促进了成骨细胞增殖;在4 d促进了早期分化指标ALP,BSP和cbfa-1 mRNA的表达;在21d促进了晚期分化指标OC和OPN mRNA的表达.与此同时,La3 使成骨细胞骨架发生重组.另外,Western Blot分析证实La3 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,La3 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.     

骨碎补有效成分柚皮甙对人骨髓间充质干细胞的影响

目的探讨骨碎补的有效成分枘皮甙对人骨髓间充质于细胞（hMSCs）增殖、分化的影响。方法将骨碎补的有效成分柚皮甙以及成骨诱导液（地塞米松、维生素C、β-甘油磷酸钠）与hMSCs共同体外培养，用倒置显微镜观察细胞生长情况、CCK-8法检测细胞的毒性和增殖作用、碱性磷酸酶（ALP）活性测定、von kossa钙结节染色。结果50μg／L柚皮甙对细胞无毒性、能促进hMSCs增殖，碱性磷酸酶（ALP）活性、von kossa钙结节染色与卒白对照组比较有明硅差异（P〈0．05）。结论柚皮甙对hMSCs有保护作用并能促进其增殖、分化。     

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.     

钽微粒对人成骨细胞分化影响的实验

磨损颗粒是假体周围骨溶解,直接影响假体使用寿命的主要原因.取人工髋关节置换中截除的松质骨提取成骨细胞,分别加入常规培养基、含钽(tantalum,Ta)微粒悬液培养基、含钛(titanium,Ti)微粒悬液培养基混合培养.观察微粒对共培养的人成骨细胞的增殖分化过程的影响.结果显示Ta微粒和Ti微粒样品内毒素含量符合USP规定,常规培养基组细胞液内碱性磷酸酶(alkaline phosphatase,ALP)、骨钙素(osteocalcin,OC)、Ⅰ型胶原(type I collagen,ColⅠ)和羟脯氨酸(hydroxyproline,HYP)含量最高,Ta微粒组次之,Ti微粒组最低(p<0.05).Real-time PCR检测结果显示各组核心结合因子α1(core-binding factorα1,Cbfa1)、ColⅠ、Osterix(OSX)基因mRNA的相对表达水平从高到低依次为常规培养基组、Ta微粒组、Ti微粒组(p<0.05).以上指标正常组均随天数的增加表达水平逐渐增强;而Ta微粒组有所升高,Ti微粒组则有所下降.提示与Ti微粒相比,Ta微粒对人成骨细胞的分化无明显的抑制作用.     

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.     

3-MA 对阿霉素诱导 K562、K562 / ADM 细胞凋亡及其相关基因 mRNA 表达的影响

目的：通过自噬抑制剂3-甲基腺嘌呤（3-MA）对阿霉素（ADM）诱导白血病K562细胞及K562／ADM细胞的细胞效应和自噬基因Beclinl、凋亡抑制基因SurvivinmR．NA表达的变化观察,探讨自噬在细胞凋亡中的作用和机制．方法：体外培养K562和K562／ADM细胞,采用MTT法分别检测ADM及3-MA预处理对K562、K562／ADM细胞增殖的影响,流式细胞仪检测细胞凋亡率,实时RT—PCR法检测细胞自噬及凋亡相关基因（Beclinl、Survivin）mRNA表达的变化．结果：ADM可抑制K562与K562／ADM细胞增殖,且抑制作用呈现浓度与时间依赖性．ADM诱导组K562与K562／ADM细胞在24h、48h、72h细胞凋亡率均较空白对照组明显提高（P〈0．05）．在ADM诱导前,经3-MA预处理可使ADM诱导的K562与K562／ADM细胞抑制率和细胞凋亡率均较单用ADM显著提高（尸〈0．05）,Bedin1、SurvivinmRNA相对表达量均较单用ADM明显下降（P〈0．05）,呈正相关（r=0．827,P〈0．01）．结论：ADM可抑制K562、K562／ADM细胞的生长,并诱导细胞凋亡．3-MA通过抑制细胞的自噬可增强ADM诱导白血病细胞K562、K562／ADM的凋亡,其机制可能与下调BeclinlmRNA表达,而使Survivin表达受抑制有关．     

AP-1及其相关基因uPA、uPAR在肺癌中的表达及其意义

研究AP-1及其相关基因uPA、uPAR在人肺癌中的表达及其与肺癌临床病理特征的关系.通过免疫组织化学方法检测101例肺癌组织及7例正常肺组织中AP-1、uPA、uPAR的表达情况,利用CMIAS2000型多功能真彩病理图像分析系统测量计算各病例中c-jun、c-fos、uPA、uPAR蛋白阳性细胞的平均光密度(DAO)和积分光密度(DIO).c-jun、c-fos在肺癌组织中表达的阳性率及其共表达率均显著高于正常肺组织;二者在NSCLC的表达显著强于SCLC;不同组织学类型肺癌中二者表达具有显著性差异,腺癌中表达最强,小细胞肺癌中表达最弱;c-jun、c-fos与肺癌分级、合并分期、淋巴结转移均呈显著正相关.uPA、uPAR与肺癌淋巴结转移密切相关,与合并分期呈显著正相关;uPAR与肺癌分级呈显著正相关;各指标之间均存在显著正相关关系.研究结果表明uPA、uPAR参与了肺癌侵袭转移,AP-1信号传导通路在该过程中起了重要调节作用.     

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.     

Abrogation by c-myc of G1 phase arrest induced by RB protein but not by p53.

Inactivating mutations of the retinoblastoma gene (RB) are found in a wide variety of tumour cells. Replacement of wild-type RB can suppress the tumorigenicity of some of these cells, suggesting that the RB protein (Rb) may negatively regulate cell growth. As activation of c-myc expression promotes cell proliferation and blocks differentiation, it may positively regulate cell growth. The c-myc protein is localized in the nucleus and can physically associate with RB protein in vitro, hence c-myc may functionally antagonize RB function. Microinjection of Rb in G1 phase reversibly arrests cell-cycle progression. Here we co-inject RB protein with c-myc, EJ-ras, c-fos or c-jun protein. Co-injection of c-myc, but not EJ-ras, c-fos or c-jun, inhibits the ability of Rb to arrest the cell cycle. The c-myc does not inhibit the activity of another tumour supressor, p53 (ref. 12). Thus, c-myc and RB specifically antagonize one another in the cell.     

拟南芥一氧化氮相关突变体在干旱胁迫下抗旱相关基因表达的研究

据报道,拟南芥一氧化氮（nitric oxide,NO）合成缺陷突变体noa1较野生型Col-0抗旱,而个别抗旱相关基因表达的增强是其抗旱机理之一.为了证实该结果及研究NO信号分子在抗旱中的作用,系统地对8种共12个不同类型的抗旱相关基因在干旱处理不同时间的拟南芥野生型和noa1及亚硝基谷胱甘肽还原酶功能缺失突变体gsnor1-3间的表达情况进行了反转录PCR分析.研究结果表明,抗旱相关基因的表达在拟南芥不同基因型间无明显差异,noa1的耐旱性与抗旱相关基因的表达无显著相关性.     

Electrospun Nanofibers of Hydroxyapatite/Collagen/Chitosan Promote Osteogenic Differentiation of BMSCs

Bone tissue engineering, aiming at developing bone substitutes for repair and regeneration of bone defects instead of using autologous bone grafts, has attracted wide attention in the field of tissue engineering and regenerative medicine. Developing biomimetic biomaterial scaffolds able to regulate osteogenic differentiation of stem cells could be a promising strategy to improve the therapeutic efficacy. In this study, clectrospun composite nanofibers of hydroxyapatite/collagen/chitosan （ HAp/Col/CTS ） resembling the fibrous nanostructure and constituents of the hierarchically organized natural bone, were prepared to investigate their capacity for promoting bone mesenchymal stem cells （BMSCs） to differentiate into the osteogenic lineage in the absence and presence of the osteogenlc supplementation, respectively. Call morphology, proliferation and quantified specific osteogenic protein expression on the electrospun HAp/Coi/CTS scaffolds were evaluated in comparison with different controls including dectrospun nanofibrous CTS, HAp/CTS and tissue culture plate. Our remits showed that the nanofibrous HAp/Col/CTS scaffolds supported better spreading and proliferation of the BMSCs than other substrates （ P 〈 0.01 ）. Expressions of osteogenesis protein markers, alkaline phosphatase （ALP） and Col, were significantly upregulated on the HAp/Col/CTS than those on the CTS （P 〈0.01） and HAp/ CTS （P 〈 0. 05 ） scaffolds in the absence of the osteogeulc supplementation. Moreover, presence of osteogeulc supplementation also proved to enhance osteogeule differentiation of BMSCs on HAp/ Col/CTS scaffolds, indicative of a synergistic effect. This study highlights the potential of BMSCs/HAp/Col/CTS cell-scaffold system for functional bone repair and regeneration applications.