Notch2胞内段基因过表达对K562细胞增殖的影响及其机制

目的探讨外源性Notch2胞内段基因过表达对慢性粒细胞白血病(Chronic myeloid leukemia,CML)细胞株K562增殖的影响及其机制。方法将携带Notch2胞内段(ICN2)基因的质粒转染K562细胞,MTT法检测细胞的增殖;流式细胞仪检测细胞的细胞周期分布;RT-PCR检测Notch2基因全长mRNA的转录,Western blot检测Notch2蛋白的表达;RT-PCR检测Notch通路下游靶基因Hes1、Hey1及细胞增殖、凋亡相关基因numb、Bcl-2、NF-κB和TGF-β1的mRNA转录水平。结果与未转染组相比,转染组K562细胞数量减少,增殖显著受抑(P<0.01);转染48 h后的K562细胞G1期细胞比例显著增多(P<0.01),S期细胞比例显著减少(P<0.01);Notch2基因mRNA及下游靶基因Hes1和Hey1 mRNA转录水平均明显增强(P<0.01),Notch2蛋白表达量增加(P<0.01);numb基因mRNA转录水平无变化;Bcl-2表达下调,NF-κB和TGF-β1表达上调。结论Notch2胞内段基因过表达可抑制K562细胞增殖,其机制可能是通过上调TGF-β1和NF-κB基因表达及下调Bcl-2基因表达,将细胞阻滞于G1期来实现的。     

细胞传代对骨髓间充质干细胞成软骨细胞分化的影响

目的 观察在成软骨诱导培养条件下,细胞传代对骨髓间充质干细胞(MSCs)体外成软骨能力的影响.方法 不同代MSCs成软骨诱导后,观察细胞生物学特性以及通过免疫荧光,RT-PCR测定特异性软骨细胞外基质aggrecan的表达情况.结果 经成软骨诱导后,第2、4代MSCs表达aggrecan明显较第6、8代细胞高.结论 MSCs很可能由多种形态功能接近,分化潜能有略有差异的细胞组成;在成软骨诱导培养条件下,对此传代后成软骨能力减弱.     

金雀异黄酮对脂多糖处理的软骨细胞增殖抑制的拮抗及抗炎作用

目的观察金雀异黄酮(Genistein,GEN)对脂多糖(Lipopolysaccharides,LPS)处理的软骨细胞增殖抑制的拮抗及抗炎作用。方法体外培养新西兰兔关节软骨细胞,将细胞分为正常对照组、LPS单独处理组(10μg/ml)及LPS(10μg/ml)+不同剂量GEN(1、5、10、15μg/ml)处理组,采用MTT法检测各组细胞的增殖活性,RT-PCR法检测各组细胞相关炎性因子IL-1β、诱导型一氧化氮合酶iNOS、转录调节因子p53基因mRNA的表达。结果剂量为1μg/ml的GEN能拮抗软骨细胞经LPS处理后引起的增殖活性降低,且能抑制经LPS处理后软骨细胞IL-1β、iNOS、p53基因mRNA的表达。结论 GEN对LPS处理的软骨细胞可能具有拮抗其增殖抑制及抗炎的作用。     

香草乙酮对胶原蛋白COL17A1基因表达的影响及在护肤品中的应用

研究香草乙酮对17型胶原蛋白(COL17A1)基因表达的"量效关系"和安全剂量,在此基础上开发新型抗衰老护肤产品并检验实际功效。体外培养HaCaT细胞(人永生化角质形成细胞株),用不同浓度的香草乙酮处理细胞,实时定量PCR法(RT-PCR)检测HaCaT细胞中COL17A1基因mRNA的转录水平,Western blot检测COL17A1蛋白表达水平;利用鸡胚绒毛膜尿囊膜血管毒性实验(CAMVA)确定既安全又有效诱导COL17A1表达的香草乙酮浓度制备护肤品,并在28名中老年志愿者皮肤上进行功效测实。结果表明,经香草乙酮处理后,HaCaT细胞中COL17A1基因表达量在一定浓度范围内显著提高,当香草乙酮浓度达到60 μmol/L时,COL17A1基因表达量最高;浓度为120 μmol/L时,COL17A1基因表达量开始呈下降趋势。香草乙酮浓度小于8 400 μmol/L时具有良好安全性;人体皮肤皱纹严重程度在使用香草乙酮后得到一定缓解。     

激活素受体相互作用蛋白1,2在小鼠脑神经细胞中的共表达

目的探讨激活素受体相互作用蛋白1(activin receptor-interaction protein 1,ARIP1)和ARIP2在小鼠脑神经细胞中的共表达。方法 RT-PCR法检测C57BL小鼠小脑、垂体、大脑、脾脏、心脏、肾脏、肝脏、胰腺组织中ARIP1和ARIP2基因mRNA的转录;以融合蛋白GST-ARIP1C和MBP-ARIP2C作为抗原免疫新西兰白兔,制备抗ARIP1和ARIP2抗体,经A蛋白亲和层析柱纯化后,ELISA法检测抗体的交叉反应;用制备的抗体,采用免疫组织化学染色检测ARIP1和ARIP2在小鼠脑组织中的表达;RT-PCR法检测小鼠神经母细胞瘤Neuro-2a细胞中激活素ⅡA型受体(activin receptorⅡA,ActRⅡA)、ARIP1和ARIP2基因mRNA的转录。结果在小鼠大脑、小脑及垂体组织中可见ARIP1基因mRNA的转录,在小鼠各部位组织中均可见ARIP2基因mRNA的转录;制备的兔抗ARIP1和ARIP2抗体无交叉反应;ARIP1和ARIP2在小鼠脑组织的海马和下丘脑同时表达;ActRIⅡA及ARIP1和ARIP2 mRNA在神经元样细胞系Neuro-2a细胞中共表达。结论 ARIP1和ARIP2可在小鼠脑神经细胞中共表达。     

β-catenin对骨形态发生蛋白9诱导间充质干细胞成骨分化的影响

目的探讨经典Wnt信号通路关键节点β-catenin对骨形态发生蛋白9(bone morphogenetic protein 9,BMP9)诱导间充质干细胞(mesenchymal stem cells,MSCs)成骨分化的影响。方法用重组腺病毒介导BMP9在C3H10T1/2细胞中过表达,联用β-catenin重组腺病毒上调β-catenin的表达,并通过RNA干扰抑制β-catenin的表达。分析C3H10T1/2细胞碱性磷酸酶(alkaline phosphatase,ALP)活性的变化;RT-PCR检测细胞成骨分化相关基因骨桥蛋白(osteopontin,OPN)和骨钙蛋白(osteocalcin,OC)基因mRNA的转录水平;茜素红S染色检测细胞的钙盐沉积。结果 BMP9单独作用能诱导C3H10T1/2细胞向成骨方向分化,并增强细胞ALP活性;单独的β-catenin无成骨诱导作用,但可剂量依赖性地增强BMP9诱导的C3H10T1/2细胞的ALP活性,并促进BMP9诱导的细胞OPN和OC基因mRNA的转录水平及钙盐沉积;抑制β-catenin表达可显著降低BMP9诱导的C3H1OT1/2细胞的ALP活性(P0.05),下调OPN和OC基因mRNA的转录水平,并抑制钙盐沉积。结论经典Wnt信号通路可能通过β-catenin协同BMP9诱导C3H10T1/2细胞成骨分化,且BMP9诱导的成骨分化可能需要通过Wnt/β-catenin途径来实现。     

4种来源的间充质干细胞的生物学特性比较

目的比较骨髓、骨骼肌、肋软骨膜及软骨来源的间充质干细胞(mesenchymal stem cells,MSCs)的生长、免疫表型及体外诱导分化能力的差异。方法分别获取新西兰大白兔的骨髓、骨骼肌、肋软骨膜细胞及软骨细胞,体外培养观察其生长形态、表型特征及其诱导成骨、成脂的分化能力。结果 4种来源的MSCs均从第2代开始转变为梭形,第3代逐渐出现漩涡状排列;骨髓、骨骼肌和软骨膜来源的细胞生长状态良好,可传10代以上,但软骨来源的细胞在第5代后,逐渐退变死亡。骨髓来源的原代细胞部分表达CD29、CD90、CD34,传代后不表达CD34,但CD29、CD90的表达逐代增强;骨骼肌、软骨膜及软骨来源的原代细胞均不表达CD29、CD90和CD34,从第2代开始均表达CD29、CD90,且不断增强;仅原代软骨细胞表达Ⅱ型胶原,至第2代基本不表达;无细胞表达Desmin。4种来源的第3代细胞经诱导均可分化为成骨细胞或成脂细胞。结论 4种来源的干细胞均具有MSCs的特点,但骨髓、骨骼肌、软骨膜来源的干细胞的生长明显强于软骨来源的干细胞。     

Notch1在隐睾SD幼鼠睾丸组织中的表达及其意义

目的探讨环境内分泌干扰物邻苯二甲酸二(2-乙基)已酯(Di-2-ethylhexy1 phthalate,DEHP)作用于SD孕鼠后,对哺乳期雄性幼鼠睾丸组织中Notch1的表达及其在隐睾所致不育症中可能发挥的作用。方法将30只妊娠第12.5天的SD孕鼠随机分为正常对照组、玉米油对照组和DEHP诱导隐睾组,每组10只。玉米油对照组和DEHP诱导隐睾组SD孕鼠自妊娠第12.5～19.5天,分别每日灌胃玉米油(2 ml)和DEHP(500 mg/kg),正常对照组不给药。取各组出生后第20天的雄性幼鼠,镜下观察睾丸位置,并统计隐睾发生情况。采用RT-PCR及Western blot法检测各组雄性幼鼠睾丸组织中Notch1基因mRNA和蛋白的表达水平。结果与正常对照组和玉米油对照组相比,DEHP诱导隐睾组雄性幼鼠的隐睾发生率明显上升(P<0.05);DEHP诱导隐睾组雄性幼鼠睾丸组织中Notch1基因mRNA和蛋白表达水平均明显低于正常对照组和玉米油对照组(P<0.05)。结论孕期暴露环境内分泌干扰物DEHP可引起雄性子代发生隐睾,隐睾子代睾丸组织中Notch1表达有改变,并可能与隐睾导致的不育有关。     

淫羊藿苷调控BMP-2与OSX促进成骨细胞增殖的研究

目的:探讨淫羊藿苷(ICA)调控BMP-2与OSX对成骨细胞MC3T3-E1增殖的影响。方法:不同浓度淫羊藿苷处理成骨细胞后,细胞增殖实验(CCK-8)和碱性磷酸酶(ALP)检测评价成骨增殖分化能力,激光共聚焦显微镜观察成骨细胞的骨架形态,免疫印迹法(WesternBlot)检测骨形态发生蛋白-2(BMP-2)和成骨相关转录因子(OSX)表达。结果:与对照组相比,淫羊藿苷促进成骨细胞增殖分化,4μg/L淫羊藿苷对成骨细胞增殖作用最佳。细胞活性升高,镜下可见细胞伸展更多伪足,Western Blot显示BMP-2和OSX表达增高(p0.05)。结论:淫羊藿苷有促进成骨细胞增殖分化,可能是通过调节BMP-2和OSX蛋白表达。     

人真皮成纤维细胞受长波紫外线辐照引起基因表达动态变化

通过不同累积剂量(1～7天)长波紫外线UVA辐照人原代真皮成纤维细胞,研究其细胞外基质、细胞周期及衰老相关基因动态变化情况.结果显示,胶原蛋白基因COL1A2、COL4A1在辐照1天时表达上调,辐照3天后下降;而弹性蛋白基因ELN在辐照1天时表达不变,辐照3天后下降;基质金属蛋白酶基因MMP1、MMP9在辐照5天时表达...     

Regenerative Potential of Platelet Concentrate Lysate in Mechanically Injured Cartilage and Matrix-Associated Chondrocyte Implantation In Vitro

Adjuvant therapy in autologous chondrocyte implantation (ACI) can control the post-traumatic environment and guide graft maturation to support cartilage repair. To investigate both aspects, we examined potential chondro-regenerative effects of lysed platelet concentrate (PC) and supplementary interleukin 10 (IL-10) on mechanically injured cartilage and on clinically used ACI scaffolds. ACI remnants and human cartilage explants, which were applied to an uniaxial unconfined compression as injury model, were treated with human IL-10 and/or PC from thrombocyte concentrates. We analyzed nuclear blebbing/TUNEL, sGAG content, immunohistochemistry, and the expression of COL1A1, COL2A1, COL10A1, SOX9, and ACAN. Post-injuriously, PC was associated with less cell death, increased COL2A1 expression, and decreased COL10A1 expression and, interestingly, the combination with Il-10 or Il-10 alone had no additional effects, except on COL10A1, which was most effectively decreased by the combination of PC and Il-10. The expression of COL2A1 or SOX9 was statistically not modulated by these substances. In contrast, in chondrocytes in ACI grafts the combination of PC and IL-10 had the most pronounced effects on all parameters except ACAN. Thus, using adjuvants such as PC and IL-10, preferably in combination, is a promising strategy for enhancing repair and graft maturation of autologous transplanted chondrocytes after cartilage injury.     

Pleiotropic Roles of NOTCH1 Signaling in the Loss of Maturational Arrest of Human Osteoarthritic Chondrocytes

Notch signaling has been identified as a critical regulator of cartilage development and homeostasis. Its pivotal role was established by both several joint specific Notch signaling loss of function mouse models and transient or sustained overexpression. NOTCH1 is the most abundantly expressed NOTCH receptors in normal cartilage and its expression increases in osteoarthritis (OA), when chondrocytes exit from their healthy “maturation arrested state” and resume their natural route of proliferation, hypertrophy, and terminal differentiation. The latter are hallmarks of OA that are easily evaluated in vitro in 2-D or 3-D culture models. The aim of our study was to investigate the effect of NOTCH1 knockdown on proliferation (cell count and Picogreen mediated DNA quantification), cell cycle (flow cytometry), hypertrophy (gene and protein expression of key markers such as RUNX2 and MMP-13), and terminal differentiation (viability measured in 3-D cultures by luminescence assay) of human OA chondrocytes. NOTCH1 silencing of OA chondrocytes yielded a healthier phenotype in both 2-D (reduced proliferation) and 3-D with evidence of decreased hypertrophy (reduced expression of RUNX2 and MMP-13) and terminal differentiation (increased viability). This demonstrates that NOTCH1 is a convenient therapeutic target to attenuate OA progression.     

Gremlin-1 and BMP-4 Overexpressed in Osteoarthritis Drive an Osteochondral-Remodeling Program in Osteoblasts and Hypertrophic Chondrocytes

Osteoarthritis (OA) is a whole joint disease characterized by an important remodeling of the osteochondral junction. It includes cartilage mineralization due to chondrocyte hypertrophic differentiation and bone sclerosis. Here, we investigated whether gremlin-1 (Grem-1) and its BMP partners could be involved in the remodeling events of the osteochondral junction in OA. We found that Grem-1, BMP-2, and BMP-4 immunostaining was detected in chondrocytes from the deep layer of cartilage and in subchondral bone of knee OA patients, and was positively correlated with cartilage damage. ELISA assays showed that bone released more Grem-1 and BMP-4 than cartilage, which released more BMP-2. In vitro experiments evidenced that compression stimulated the expression and the release of Grem-1 and BMP-4 by osteoblasts. Grem-1 was also overexpressed during the prehypertrophic to hypertrophic differentiation of murine articular chondrocytes. Recombinant Grem-1 stimulated Mmp-3 and Mmp-13 expression in murine chondrocytes and osteoblasts, whereas recombinant BMP-4 stimulated the expression of genes associated with angiogenesis (Angptl4 and osteoclastogenesis (Rankl and Ccl2). In conclusion, Grem-1 and BMP-4, whose expression at the osteochondral junction increased with OA progression, may favor the pathological remodeling of the osteochondral junction by inducing a catabolic and tissue remodeling program in hypertrophic chondrocytes and osteoblasts.     

Molecular Actions of Ovarian Cancer G Protein-Coupled Receptor 1 Caused by Extracellular Acidification in Bone

Extracellular acidification occurs under physiologic and pathologic conditions, such as exercise, ischemia, and inflammation. It has been shown that acidosis has various adverse effects on bone. In recent years there has been increasing evidence which indicates that ovarian cancer G protein-coupled receptor 1 (OGR1) is a pH-sensing receptor and mediates a variety of extracellular acidification-induced actions on bone cells and other cell types. Recent studies have shown that OGR1 is involved in the regulation of osteoclast differentiation, survival, and function, as well as osteoblast differentiation and bone formation. Moreover, OGR1 also regulates acid-induced apoptosis of endplate chondrocytes in intervertebral discs. These observations demonstrate the importance of OGR1 in skeletal development and metabolism. Here, we provide an overview of OGR1 regulation ofosteoclasts, osteoblasts, and chondrocytes, and the molecular actions of OGR1 induced by extracellular acidification in the maintenance of bone health.     

BMP3 Alone and Together with TGF-β Promote the Differentiation of Human Mesenchymal Stem Cells into a Nucleus Pulposus-Like Phenotype

Human mesenchymal stem cells (MSCs) have the potential to differentiate into nucleus pulposus (NP)-like cells under specific stimulatory conditions. Thus far, the effects of bone morphogenetic protein 3 (BMP3) and the cocktail effects of BMP3 and transforming growth factor (TGF)-β on MSC proliferation and differentiation remain obscure. Therefore, this study was designed to clarify these unknowns. MSCs were cultured with various gradients of BMP3 and BMP3/TGF-β, and compared with cultures in basal and TGF-β media. Cell proliferation, glycosaminoglycan (GAG) content, gene expression, and signaling proteins were measured to assess the effects of BMP3 and BMP3/TGF-β on MSCs. Cell number and GAG content increased upon the addition of BMP3 in a dose-dependent manner. The expression of COL2A1, ACAN, SOX9, and KRT19 increased following induction with BMP3 and TGF-β, in contrast to that of COL1A1, ALP, OPN, and COMP. Smad3 phosphorylation was upregulated by BMP3 and TGF-β, but BMP3 did not affect the phosphorylation of extracellular-signal regulated kinase (ERK) 1/2 or c-Jun N-terminal kinase (JNK). Our results reveal that BMP3 enhances MSC proliferation and differentiation into NP-like cells, as indicated by increased cell numbers and specific gene expressions, and may also cooperate with TGF-β induced positive effects. These actions are likely related to the activation of TGF-β signaling pathway.     

Physosmotic Induction of Chondrogenic Maturation Is TGF-β Dependent and Enhanced by Calcineurin Inhibitor FK506

Increasing extracellular osmolarity 100 mOsm/kg above plasma level to the physiological levels for cartilage induces chondrogenic marker expression and the differentiation of chondroprogenitor cells. The calcineurin inhibitor FK506 has been reported to modulate the hypertrophic differentiation of primary chondrocytes under such conditions, but the molecular mechanism has remained unclear. We aimed at clarifying its role. Chondrocyte cell lines and primary cells were cultured under plasma osmolarity and chondrocyte-specific in situ osmolarity (+100 mOsm, physosmolarity) was increased to compare the activation of nuclear factor of activated T-cells 5 (NFAT5). The effects of osmolarity and FK506 on calcineurin activity, cell proliferation, extracellular matrix quality, and BMP- and TGF-β signaling were analyzed using biochemical, gene, and protein expression, as well as reporter and bio-assays. NFAT5 translocation was similar in chondrocyte cell lines and primary cells. High supraphysiological osmolarity compromised cell proliferation, while physosmolarity or FK506 did not, but in combination increased proteoglycan and collagen expression in chondrocytes in vitro and in situ. The expression of the TGF-β-inducible protein TGFBI, as well as chondrogenic (SOX9, Col2) and terminal differentiation markers (e.g., Col10) were affected by osmolarity. Particularly, the expression of minor collagens (e.g., Col9, Col11) was affected. The inhibition of the FK506-binding protein suggests modulation at the TGF-β receptor level, rather than calcineurin-mediated signaling, as a cause. Physiological osmolarity promotes terminal chondrogenic differentiation of progenitor cells through the sensitization of the TGF-β superfamily signaling at the type I receptor. While hyperosmolarity alone facilitates TGF-β superfamily signaling, FK506 further enhances signaling by releasing the FKBP12 break from the type I receptor to improve collagenous marker expression. Our results help explain earlier findings and potentially benefit future cell-based cartilage repair strategies.     

Impact of Musashi-1 and Musashi-2 Double Knockdown on Notch Signaling and the Pathogenesis of Endometriosis

The stem cell marker and RNA-binding protein Musashi-1 is overexpressed in endometriosis. Musashi-1-siRNA knockdown in Ishikawa cells altered the expression of stem cell related genes, such as OCT-4. To investigate the role of both human Musashi homologues (MSI-1 and MSI-2) in the pathogenesis of endometriosis, immortalized endometriotic 12-Z cells and primary endometriotic stroma cells were treated with Musashi-1- and Musashi-2-siRNA. Subsequently, the impact on cell proliferation, cell apoptosis, cell necrosis, spheroid formation, stem cell phenotype and the Notch signaling pathway was studied in vitro. Using the ENDOMET Turku Endometriosis database, the gene expression of stem cell markers and Notch signaling pathway constituents were analyzed according to localization of the endometriosis lesions. The database analysis demonstrated that expression of Musashi and Notch pathway-related genes are dysregulated in patients with endometriosis. Musashi-1/2-double-knockdown increased apoptosis and necrosis and reduced stem cell gene expression, cell proliferation, and the formation of spheroids. Musashi silencing increased the expression of the anti-proliferation mediator p21. Our findings suggest the therapeutic potential of targeting the Musashi–Notch axis. We conclude that the Musashi genes have an impact on Notch signaling and the pathogenesis of endometriosis through the downregulation of proliferation, stemness characteristics and the upregulation of apoptosis, necrosis and of the cell cycle regulator p21.     

Blocking the Function of Inflammatory Cytokines and Mediators by Using IL-10 and TGF-β: A Potential Biological Immunotherapy for Intervertebral Disc Degeneration in a Beagle Model

The debilitating effects of lower back pain are a major health issue worldwide. A variety of factors contribute to this, and oftentimes intervertebral disk degeneration (IDD) is an underlying cause of this disorder. Inflammation contributes to IDD, and inflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-1β, play key roles in the pathology of IDD. Therefore, the development of treatments that inhibit the expression and/or effects of TNF-α and IL-1β in IDD patients should be a promising therapeutic approach to consider. This study characterized the potential to suppress inflammatory cytokine production in degenerative intervertebral disc (NP) cells by treatment with IL-10 and TGF-β in a canine model of IDD. IDD was induced surgically in six male beagles, and degenerative NP cells were isolated and cultured for in vitro studies on cytokine production. Cultured degenerative NP cells were divided into four experimental treatment groups: untreated control, IL-10-treated, TGF-β-treated, and IL-10- plus TGF-β-treated cells. Cultured normal NP cells served as a control group. TNF-α expression was evaluated by fluorescence activated cell sorting (FACS) analysis and enzyme-linked immunosorbent assay (ELISA); moreover, ELISA and real-time PCR were also performed to evaluate the effect of IL-10 and TGF-β on NP cell cytokine expression in vitro. Our results demonstrated that IL-10 and TGF-β treatment suppressed the expression of IL-1β and TNF-α and inhibited the development of inflammatory responses. These data suggest that IL-10 and TGF-β should be evaluated as therapeutic approaches for the treatment of lower back pain mediated by IDD.