17β-雌二醇对体外培养人成骨细胞CTGF和PAIP-1基因表达的作用

目的　观察结缔组织生长因子 (CTGF)和多聚腺苷酸结合蛋白相互作用蛋白 1(PAIP 1)mRNA在体外培养的人成骨细胞增殖分化不同阶段的表达 ,研究 17β- 雌二醇 (E2 )对成骨细胞CTGF和PAIP 1mRNA表达的作用 ,探讨雌激素对骨组织保护作用的新机制。方法　用改良的钙 钴法ALP染色 ,vanGieson法 1型胶原染色 ,0. 1%茜素红矿化结节染色 ,半定量RT- PCR检测成骨细胞碱性磷酸酶 (ALP)、骨钙素 (OC)和 1型胶原mRNA的表达 ,半定量RT- PCR和Northernblot方法检测成骨细胞CTGF和PAIP- 1mRNA表达。结果　半定量RT -PCR和组织化学染色的结果均表明 ,成骨细胞接种培养后 0～11d为细胞增殖阶段 ,7～ 15d为骨基质成熟阶段 ,15d后为骨基质矿化阶段 ;在成骨细胞培养的不同阶段 ,均检测到CTGF和PAIP- 1mRNA的表达 ,E2 可显著下调成骨细胞CTGFmRNA的表达 ,呈时间剂量依赖关系 (P <0 . 0 1) ,但对PAIP 1mRNA表达无明显作用。结论　E2 时间剂量依赖性下调成骨细胞CTGFmRNA的表达 ,但对PAIP 1mRNA表达无明显影响。     

不同牵张应力对成骨细胞MC3T3-E1分化及Writ信号转导通路的影响研究

目的 研究不同强度和时间的机械牵张应力刺激对成骨细胞的分化及Writ信号转导通路的影响。方法 采用Flexcell- FX5000细胞加力系统,分别采用3%,6% ,12%的形变幅度的正弦波对MC3T3细胞进行牵张应力干预,0. 5 Hz的频率,时间分别为2 ,4,8 h。干预结束后分别检测细胞的ALP活性;提取细胞总RNA后采用Real time-PCR分别检测骨钙蛋白（Osteocalcin, OC)、Runx2、0slerix、Wntl、β-catenin、DKK-1 mRNA 的表达;提取细胞总蛋白后采用 Werstern blot 法分别检测 Wntl、β-catenin 和Phosphor-33/37-β-catenin的蛋白表达量。结果 3%和6%强度牵张干预后,成骨细胞ALP活性升高,OC、Runx2、0sterix、 Wntl , β-catenin mRNA表达升高,而DKK-1 mRNA表达下降。6%的效应高于3% ,且干预4 h后升高幅度最大。12%强度牵张干预后,成骨细胞的ALP活性和OC mRNA表达水平下降。Wntl、β-catenin和Phosphor-33/37-β-calenin蛋白表达水平与上述mRNA表达水平相符。结论 中小强度的牵张刺激可以上调Win信号转导通路,促进成骨细胞分化,而大强度及长时间连续干预则无成骨分化作用,甚至有抑制作用。     

人骨髓间充质干细胞经富血小板血浆诱导后的增殖与成骨的分化能力

目的 通过系统评价经富血小板血浆(PRP)诱导后的人骨髓间充质干细胞(HMSC)增殖与成骨分化能力,为PHP临床骨缺损修复应用提供更为全面的依据.方法 将单纯血清培养(FCS)与PRP诱导后的HMSC分为FCS组、PRP组(PRP诱导后的HMSC),MTT法检测细胞增殖活性;将MSC分为PRP组、FCS组、地塞米松组(PRP诱导后的HMSC加DEX组),通过ALP染色、钙盐沉积染色、RT-PCR法检测ALP、OC、Coll-Ⅰ、ON、Cbfal、TGF-β1 mRNA表达来评价成骨分化能力.结果 PRP组HMSC增殖能力同FCS组相比差异无统计学意义(P＞0.05);ALP、OC、Coll-Ⅰ、ON、Cbfal mRNA表达显示PRP组与FCS组无明显差别;同FCS组相比PRP组TGF-β1 mRNA表达增高;同PRP组、FCS组相比,PRP诱导后的HMSC经DEX作用后(DEX组),ALP阳性细胞数钙盐沉积明显增多,ALP、OC mRNA表达增高.结论 PRP诱导后的HMSC能恢复到正常的增殖速率;PRP诱导后的HMSC具有与正常的HMSC相同的成骨分化能力,在体外仍可经DEX诱导向成骨细胞分化,PRP诱导后的HMSC仍能维持较强的成骨分化活性;PRP作用后的HMSC能维持较高的TGF-β1分泌.这是PRP在体内促进骨修复的可能原因之一.     

VEGF165过表达对BMP2成骨细胞分化影响的实验研究

[目的]探讨腺病毒转染血管内皮生长因子165(vascular endothelial growth factor 165,VEGF165)对骨形态发生蛋白(bone morphogenetic protein 2,BMP2)促成骨细胞分化的抑制性作用研究。[方法]采用密度梯度离心法分离兔骨髓间充质干细胞(Bone marrow mesenchymal stem cells,BMSCs),取第3代BMSCs进行细胞表型鉴定并作为细胞实验对象,使用Ad-BMP2和Ad-BMP2-VEGF165载体体外转染BMSCs,倒置荧光显微镜下观察GFP表达变化;同时ELISA和Western blot检测BMP2和VEGF165蛋白的表达。然后,应用成骨细胞诱导培养液定向诱导BMSCs向成骨细胞分化。实验分3组:Ad-BMP2-VEGF165转染BMSCs组(Ad-BMP2-VEGF165组),AdBMP2转染BMSCs组(Ad-BMP2组),BMSCs组(对照组)。分别在成骨细胞诱导培养后7、14、21 d,通过Real-time PCR分析ALP和OC mRNA相对表达水平和碱性磷酸酶(alkaline phosphatase,ALP)活性测定、骨钙素(Osteocalcin,OC)免疫组化染色来评价各组BMSCs成骨分化潜能的影响。[结果]第3代细胞表面高表达CD29(99.82%)、CD44(94.14%),低表达CD14(3.11%)、CD34(0.34%);腺病毒转染后第5 h BMP2和h VEGF165蛋白水平表达最高;成骨细胞诱导培养14和21 d后,Ad-BMP2组成骨相关基因表达、OC免疫组化和ALP活性表达最高,结果与其他两组相比较差异具有统计学意义(P<0.05);而对照组表达最弱,与Ad-BMP2-VEGF165组比较差异具有统计学意义(P<0.05)。[结论]腺病毒载体Ad-BMP2与Ad-BMP2-VEGF165转染BMSCs后,均具有明显促进BMSCs体外诱导成骨细胞分化潜能,但Ad-BMP2诱导作用更为显著,同时说明VEGF165可能对BMSCs成骨细胞分化起抑制作用。     

骨保护蛋白基因转染成骨细胞后的表达及对其生物学行为的影响

目的 将人骨保护蛋白(OPG)基因转入体外培养的人成骨细胞,研究OPG基因在转染成骨细胞中的表达,并分析OPG基因转染对成骨细胞生物学行为的影响.方法 首先行人成骨细胞体外培养,然后用质粒peDNA3.1-hOPG转染成骨细胞,应用逆转录-聚合酶链反应(RT-PCR)和Western blot检测转染细胞OPG mRNA和蛋白质的表达.观察OPG基因转染后成骨细胞的增殖情况,对成骨细胞表达的碱性磷酸酶(ALP)和骨钙素(BGP)的含量进行检测,观察转基因细胞的生物学特性.结果 RT-PCR和Western blot检测结果表明在OPG基因转染后,成骨细胞表达的OPGmRNA和蛋白质含量均较未转染组增加.在OPG基因转染后,可明显促进成骨细胞的增殖,成骨细胞表达的ALP和BGP的含量均显著上升,而且在量效图中可以观察到随着OPG基因转染剂量增加,其增加程度也增加.结论 成骨细胞可作为转基因的受体细胞,成功表达目的 基因.转染OPG基因的成骨细胞可稳定、高效的表达OPG.OPG基因转染成骨细胞生物学特性稳定,而且可明显促进转染成骨细胞的成骨特性的表达.     

Maspin:新的潜在的骨肉瘤转移抑制基因

目的 通过对肿瘤基因组数据整体分析寻找骨肉瘤转移相关基因,初步分析其与骨肉瘤转移的关系.方法 联合分析肿瘤组织DNA拷贝数目的 变化及肿瘤转移细胞模型中表达差异基因数据,筛选骨肉瘤转移相关基因,通过RT-PCR、Westen blot及免疫组化检测该基因在不同骨肉瘤细胞系、正常成骨细胞、骨肉瘤及正常骨组织临床标本中的表达,并结合临床随访分析其与骨肉瘤患者预后的关系.结果 NCBI数据库显示18q常出现DNA拷贝数目变化和杂合性缺失.基因芯片结果 显示高转移MG63-A1细胞系与MG63wt相比142个基因表达差异在4倍以上,而在18q21区域其中一个新的肿瘤转移抑制基因Maspin在高转移MG63-A1细胞系中表达明显下降.RT-PCR、Westen blot检测证实基因芯片结果 准确,并发现Maspin在正常成骨细胞高表达,在SAOS2、143B中表达缺失.免疫组化检测显示在27例骨肉瘤临床标本中,33.3%(9/27例)的标本Maspin表达为阳性,其余为阴性;在正常骨组织中Maspin表达强阳性.Maspin表达与骨肉瘤患者预后呈正相关(P=0.032).结论 Maspin的表达随着骨肉瘤细胞系转移能力的增加而出现缺失,在骨肉瘤标本中Maspin表达与患者预后呈正相关,预示着其可能成为阻遏骨肉瘤转移的新靶点.     

RecQ5表达与骨肉瘤组织恶性程度间的关系分析

目的分析RecQ5在不同组织和细胞上的表达差异,推断RecQ5与组织恶性程度间的关系。方法选取骨肉瘤标本共35例、癌旁组织标本20例及正常骨组织标本20例,通过免疫组织化学染色法检测不同组织中RecQ5的表达情况,并进一步分析其表达程度与骨肉瘤分期的关系;培养hFOB1.19、U2OS和MG-63细胞株,通过Real-timePCR和Westernblot法检测RecQ5的表达程度。结果RecQ5在人骨肉瘤组织中低表达。正常骨组织中,RecQ5的表达阳性率为100%(20/20),在癌旁组织中的表达阳性率为90%(18/20),而在骨肉瘤组织中,RecQ5表达阳性率为68.5%(24/35);正常骨组织、癌旁组织与骨肉瘤组织的RecQ5表达阳性率之间比较差异具有统计学意义(P=0.004),但正常骨组织与癌旁组织之间的阳性率差异无统计学意义(P>0.05)。RecQ5的表达强度随着组织恶性程度的不断增加而逐渐减弱,差异具有统计学意义(P<0.05),且在骨肉瘤组织中,其也随着肿瘤分期增加而表达强度逐渐减弱(P<0.05)。RecQ5在人骨肉瘤细胞株MG-63和U2OS中mRNA和蛋白表达水平较正常人成骨细胞hFOB1.19中低,差异有统计学意义(P<0.05)。结论RecQ5的表达水平与组织的恶性程度呈负相关,RecQ5的缺失与骨肉瘤的发生存在相关性。     

富血小板血浆与地塞米松对人骨髓基质干细胞成骨分化作用的对比研究

目的 通过系统评价富血小板血浆(PRP)与地塞米松(DEX)对人骨髓基质干细胞(BMSCs)成骨分化的影响,为PRP临床骨组织修复应用提供更为可靠的实验依据.方法 将体外培养的BMSCs分为单纯血清培养组(FCS组)、PRP诱导组和DEX组,通过相差显微镜观察碱性磷酸酶(ALP)染色、钙盐沉积染色,RT-PCR法检测碱性磷酸酶(ALP)、骨钙素(OC)、Ⅰ型胶原(Coll-Ⅰ)、骨连接蛋白(ON)、中心结合因子(Cbfα1)mRNA表达系统评价PRP的成骨分化能力. 结果 PRP抑制了BMSCs向三角形、多角形细胞转变,DEX则诱导BMSCs向三角形、多角形细胞转变;PRP抑制了ALP分泌,钙盐沉积;DEX增加了ALP分泌,促进钙化结节形成.与FCS组相比,DEX促进了ALP、OCmRNA表达,PRP抑制了ALP、OC mRNA表达;PRP、DEX对Coll-Ⅰ、ON、Cbfα1 mRNA表达均无影响.结论 在本实验条件下,PRP对人BMSCs体外成骨分化的直接作用是抑制效应;在体外PRP并不能代替DEX作为人BMSCs成骨分化的诱导因子.     

牛骨胶原肽对成骨细胞分化的影响

目的研究3 mg/ml牛骨胶原肽(Bovine collagen peptides,BCP)对人成骨细胞(Human osteoblast,HOB),小鼠前成骨细胞系MC3T3分化的影响。方法 Western blot检测MC3T3细胞中BCP对Runx2表达的影响;分离培养HOB,利用对硝基苯磷酸比色法检测3 mg/ml BCP对碱性磷酸酶(Alkaline phosphatase,ALP)含量的影响;骨钙素(osteocalcin,OC)ELISA法测定BCP对OC含量的影响;茜素红矿化染色检测BCP对HOB矿化,然后用5%高氯酸进行脱色,用吸光度法检测BCP对人成骨细胞矿化程度。结果 Western blot检测表明,14 d后BCP处理的MC3T3细胞中Runx2蛋白表达水平(0.178±0.201)与CN组(0.146±0.582,P0.05)比较,有增高趋势。ALP染色结果表明,BCP混合物处理组的ALP的染色面积(33859±8221)在第10d与CN组(19900±2796)相比,显著增加(P0.05),表明BCP混合物能促进人成骨细胞早期的ALP表达量。BCP混合物处理组的OC吸光度值(0.137±0.014)在第14d高于CN组(0.086±0.023,P0.05),表明BCP混合物能促进人成骨细胞晚期阶段的OC含量。茜素红矿化染色结果表明,3mg/ml BCP能显著促进HOB的矿化骨基质的形成。当使用5%的高氯酸脱色后,BCP处理组在490 nm处测吸光度值(0.579±0.093)显著高于CN组(0.193±0.021,P0.01),表明BCP混合物能促进人成骨细胞的矿化。结论 BCP在成骨细胞分化和矿化骨基质的形成中发挥了积极作用。综合上述结果,本文为BCP混合物在骨关节炎和骨质疏松症潜在的预防和治疗提供了分子机理。     

过表达FOXO1对骨肉瘤细胞侵袭迁移能力的影响及机制研究

目的探讨FOXO1对骨肉瘤细胞侵袭迁移能力的影响及机制。方法采用实时荧光定量PCR(qRT-PCR)的方法检测人正常成骨细胞hFOB 1.19和人骨肉瘤细胞U2OS、MNNG/HOS中FOXO1的mRNA表达水平。利用pcDNA3.1-FOXO1重组质粒建立FOXO1过表达的细胞模型,空载质粒(pcDNA3.1-vector)作为对照,qRT-PCR和蛋白质印迹法(Western blot)验证转染效率。确定成功过表达FOXO1基因后利用Cell Counting Kit-8(CCK-8)实验检测骨肉瘤细胞的增殖能力(吸光度值),Transwell实验体外检测骨肉瘤细胞的侵袭和迁移能力,并采用qRT-PCR和western blot的方法检测基质金属蛋白酶9(MMP9)的表达变化。结果人骨肉瘤细胞MNNG/HOS、U2OS中FOXO1的mRNA的表达水平显著低于人成骨细胞hFOB 1.19(P0.05),且骨肉瘤细胞U2OS的FOXO1的表达水平较低。重组质粒pcDNA3.1-FOXO1转染后,骨肉瘤细胞U2OS的FOXO1表达水平显著升高。与对照组(pcDNA3.1-vector)比较,U2OS-FOXO1过表达组的细胞的增殖能力降低(P0.05),侵袭迁移和迁移能力降低,MMP9较对照组表达水平明显降低(P0.05)。结论骨肉瘤细胞中FOXO1表达水平明显低于正常成骨细胞。过表达FOXO1可能通过下调MMP9抑制骨肉瘤细胞的侵袭和迁移能力。     

Medium perfusion enhances osteogenesis by murine osteosarcoma cells in three-dimensional collagen sponges.

In this study, we examined in vitro histogenesis by murine K8 osteosarcoma cells maintained in three-dimensional (3D) collagen sponges. We tested the hypothesis that perfusion of medium enhances cell viability and their biosynthetic activity as assessed by expression of the osteoblastic phenotype and mineral deposition. At intervals, samples were harvested and analyzed histologically, biochemically, and by Northern hybridization for type I collagen, osteopontin (OPN), osteocalcin (OC), and core binding factor alpha 1 (Cbfa1). Histologic evaluation showed greater viability, more alkaline phosphatase (ALP)-positive cells, and more mineralized tissue in the perfused sponges after 21 days. Immunohistological assessment of proliferating cell nuclear antigen revealed 5-fold more proliferating cells in the perfused sponges compared with the controls (p = 0.0201). There was 3-fold more ALP activity in the perfused sponges than the controls at 6 days and 14 days (p = 0.0053). The perfused sponges contained twice the DNA and eight times more calcium than the nonperfused controls after 21 days (p < 0.0001 for both). Northern hybridization analysis revealed more mRNA for collagen type I (2-fold) and 50% more for OC at 14 days and 21 days, whereas OPN and Cbfa1 mRNA expression remained unaffected by the medium perfusion. These results show that medium perfusion had beneficial effects on the proliferation and biosynthetic activity of this osteosarcoma cell line. This system mimics the 3D geometry of bone tissue and has the potential for revealing mechanisms of regulation of osteogenesis.     

Age-dependent expression of osteoblastic phenotypic markers in normal human osteoblasts cultured long term in the presence of dexamethasone

We have previously shown that osteoblasts derived from trabecular bone explants and cultured long term in 10 nM dexamethasone ((HOB+DEX) cells) exhibited properties consistent with a more differentiated phenotype compared with those grown in the absence of dexamethasone ((HOB-DEX) cells). To characterize these two cell models further, we measured the steady-state mRNA levels of the phenotypic markers alkaline phosphatase (ALP), collagen type I (COLL) and osteocalcin (OC), OC production, and the activities of ALP and parathyroid hormone (PTH)-stimulated adenylate cyclase. These findings were then correlated with the age and sex of the bone donors. Long-term culture in dexamethasone significantly increased ALP and OC mRNA levels and the activities of ALP and PTH-stimulated adenylate cyclase but not OC production, in (HOB+DEX) compared with (HOB-DEX) cells (p<0.05). When the data were examined with respect to the age of the bone donor, age-dependent differences in the expression and responses to dexamethasone were apparent. ALP and PTH-stimulated adenylate cyclase activities decreased with increasing age of the bone donor in (HOB-DEX) and (HOB+DEX) cells (p<0.05). There were no significant correlations between phenotypic marker mRNA levels and bone donor age in (HOB-DEX) and (HOB+DEX) cells. All age-dependent decreases in ALP and PTH-stimulated cyclase activities were enhanced in the (HOB+DEX) cells. However, when the data were examined according to the sex of the bone donor, there were no differences in mRNA levels, OC production, or ALP and cyclase activities between cells from male and female donors. These results indicate an age dependence in the expression of osteoblastic markers in human bone cells at different stages of differentiation: thus osteoblastic cultures derived from older donors are likely to contain fewer osteoprogenitor cells, lower levels of glucocorticoid receptors or represent more differentiated osteblasts compared with those derived from younger donors.     

Osteogenic phenotype expression of allogeneic rat marrow cells in porous hydroxyapatite ceramics.

Porous hydroxyapatite (HA) ceramics were combined with either allogeneic (ACI) or isogeneic (Fischer 344) rat marrow cells and implanted in subcutaneous sites of Fischer rats. FK506 as an immunosuppressant or saline was administered to the recipient rats. The implanted marrow/HA composites were harvested on day 28 and analyzed for bone-forming capability by determining osteoblastic phenotype expression levels of protein synthesis and gene expression. The alkaline phosphatase (ALP) activity and osteocalcin (OC) contents were very low and mRNAs (Northern blot analysis) were not detected in the allografts without FK506. However, high activity of ALP and high content of OC were found and mRNAs were detected in the allografts with FK506 and in the isografts (with and without FK506). This analysis indicates the osteogenic potential of allogeneic marrow cells in the presence of FK506. The histologic sections revealed that allografts without FK506 did not show bone formation but did show the infiltration of many small cells in the ceramics indicating an immunologic reaction, however, the allografts with FK506 and the isografts (with and without FK506) showed consistent de novo bone formation on the HA pore surface. These results indicate that FK506 can suppress the immunologic reaction in the allografts and induce a favorable conditions to support osteoblastic differentiation of allogeneic rat marrow stromal stem cells on the surface of HA ceramics. Therefore, our study suggests the feasibility of clinical transplantation of allogeneic bone marrow for a selected bone graft in applications using adjuvant systemic immunosuppression.     

体外诱导家猪骨髓间充质干细胞向成骨细胞分化

目的观察家猪骨髓间充质干细胞(marrow mesenchymal stem cells,MSCs)在体外诱导条件下成骨分化的特征及相关基因的表达。方法选取3月龄雌性长白猪6头,无菌条件下于胫骨近端抽取骨髓15ml。采用密度梯度离心法并根据细胞贴壁特性对MSCs进行分离、纯化,倒置相差显微镜观察原代细胞生长情况,于培养第7天计算MSCs百分比含量及群体倍增值。将第1代细胞置于含1×10-8mmol/L地塞米松(dexamethasone,Dex)、10mmol/Lβ-磷酸甘油(β-glycerophosphate,β-GP)和82μg/ml抗坏血酸(ascorbic acid,Asc)的成骨诱导培养液中培养21d,作为实验组;DMEM培养液中培养作为对照组。分别行细胞形态学观察、碱性磷酸酶(alkaline phosphatase,ALP)组织化学染色、钙沉积和细胞增殖测定,采用实时定量PCR分析成骨分化的相关基因表达。结果原代MSCs特征:培养第1天,有核细胞大部分由悬浮的圆形血源性细胞组成;第3天换液弃除非贴壁细胞,MSCs克隆开始形成,细胞呈成纤维细胞样生长;第7天,镜下观察到大小不一的克隆。细胞在培养后12～14d基本长满,原代细胞群体倍增值平均为13。MSCs成骨分化:诱导培养14d实验组细胞形态由成纤维细胞样变成立方体样,而对照组细胞始终保持成纤维细胞样。培养5d,对照组细胞计数为11723±4040,实验组为10276±5513,二者差异无统计学意义(P>0.05)。与对照组相比,实验组诱导培养14d,ALP染色呈强阳性,21d钙沉积明显增加(P<0.01)。实验组MSCs成骨相关基因:核心结合因子α1(corebinding factorα1,Cbfα1)、osterix、ALP、型胶原、骨连接素(osteonectin,ON)、骨钙素(osteocalcin,OC)表达逐渐增强;Cbfα1、ALP、ON在分化早期增加明显;与第7天比较,第21天osterix和OC基因表达明显上调(P<0.05);第14天,型胶原表达也上调(P<0.05)。结论密度梯度离心法分离的猪MSCs,在体外诱导条件下能通过上调分化特异基因表达向成骨细胞分化。     

Effects of androgens on subpopulations of the human osteosarcoma cell line SaOS2

Previously, we showed that androgens stimulate murine and human osteoblast-like cell proliferation and differentiation by mechanisms involving increased responses to mitogenic growth factors (GF) and increased GF production. To explain this dual action of androgens on primary osteoblastic cell populations we advanced the hypothesis that androgens exert differential effects on osteoblastic subpopulations. We subcloned a human osteosarcoma cell line (SaOS2) into subpopulations expressing high (HAS) and low (LAS) levels of alkaline phosphatase (ALP). The obtained subclones differed significantly in their ALP production and expressed a high and low ALP phenotype, respectively, for the entire experimental period. Dihydrotestosterone (DHT) increased specific ALP activity and type-I procollagen peptide secretion in both HAS and LAS. DHT pretreatment enhanced the mitogenic action of basic fibroblast growth factor (bFGF) and insulinlike growth factor 2 (IGF2) only in HAS. The enhanced mitogenic effect of IGF2 in HAS after DHT pretreatment was associated with increased IGF2-receptor mRNA levels. Therefore, we conclude that androgens exert their osteoanabolic action (1) by stimulating differentiated functions of osteoblastic cells with a high and a low ALP phenotype, and (2) via increased growth factor receptor expression and thereby enhancing mitogenic growth factor responses only in HAS. This paper is dedicated to the occasion of Prof. Dr. R. Ziegler's 60th birthday     

Establishment and characterization of cell lines derived from a human osteosarcoma

Continuously growing cell lines have been established in vitro from a human osteosarcoma after transplantation into athymic nude mice. These cell lines grew as an adherent monolayer and consisted of various types of cells. Twelve clones were colonially isolated from a cell line, HuO-3N1, and subdivided into three groups depending on the morphologic features. The cells of HuO-3N1 and its clones had alkaline phosphatase (ALP)-positive granules in the cytoplasm. Cytogenetic studies showed that these cell lines were human aneuploid lines. A tumor was produced by injection of HuO-3N1 cells into an athymic nude mouse. ALP activity increased in a clonal cell line, HuO-3N1 cl-2, when cells were treated with 1,25-dihydroxy-vitamin D3. The proliferation of cells was inhibited when the cells were cultured in a medium supplemented with L-homoarginine, which is an inhibitor of bone and liver-specific ALP. This cell line has an osteoblastic phenotype and provides a useful model for studies of human osteosarcoma and phenotypical expression of human osteoblastic cells.     

Modulation of thrombospondin gene expression during osteoblast differentiation in MC3T3-E1 cells.

The levels of expression of two related extracellular matrix protein genes, thrombospondins 1 and 2 (TSP1 and TSP2), were analyzed in the mouse osteogenic cell line, MC3T3-E1. To monitor differentiation, we also measured two potential markers of the osteoblastic phenotype, alkaline phosphatase (ALP) activity, and alpha 1(I) collagen mRNA levels. TSP1 mRNA levels increased 10- to 15-fold during the first nine days of osteoblastic conversion, and then dropped to a level still significantly above baseline values. This increase in TSP1 mRNA closely paralleled that observed in ALP activity. In contrast, TSP2 mRNA levels were unchanged throughout the 21-day time course. These findings suggest that TSP1 is a marker for osteoblast differentiation and could play a role in the cellular changes that accompany acquisition of the osteoblastic phenotype in MC3T3-E1 cells.     

成骨细胞相关基因在软骨肉瘤中的表达

目的 通过对软骨肉瘤细胞中成骨细胞相关基因的表达进行分析,探讨软骨肉瘤中细胞分化的分子表型。方法 采用RT-PCR和印记方法半定量测定32例软骨肉瘤患者骨钙素（OC）、碱性磷酸酶（AP）、骨结合蛋白（ON）和Cbfa1的表达,分析其与组织学分级和预后的关系。结果 病理学分级与OC、AP或Cbfa1表达有明显相关性;成骨细胞相关基因在Ⅲ级软骨肉瘤的表达最为多见,骨钙素mRNA表达的检测,在12例Ⅲ级患者有9例可测到（75%）,在20例Ⅰ-Ⅱ级患者中有4例可测到（20%）（P=0.008)。在32例患者中,21个可测到APmRNA,其中12例Ⅲ级患者中有11例（92%）、20例Ⅰ-Ⅱ级患者中有10例（50%）可测到AP的表达（P=0.018)。OC或AP表达常与无病生存率下降相联系（OC：P=0.016;AP:P=0.018).CbfalmRNA只在4例Ⅲ级软骨肉瘤中测到。所有软骨肉瘤中均可测到ON,表明其表达与病理分级及无病生存率无相关性。结论 绝大多数Ⅲ级软骨肉瘤中可测到OC的表达,而在Ⅰ-Ⅱ级软骨肉瘤中只有少数表达,并且OC的表达常与预后差相关联;APmRNA阳性率在Ⅰ-打级和Ⅲ级中有明显的差别,AP的表达也和无病生存率下降呈正相关,在软骨肉瘤中OC或AP的表达可做为独立的预后指标;Cbfa1在分级高的软骨肉瘤中较常表达,表现出与OC有相同的特性。