人参皂苷Rg1协同rhBMP-2对人牙髓干细胞成牙分化作用研究

目的：探讨不同浓度人参皂苷Rg1、重组骨形态发生蛋白2（rhBMP-2）及两者联合应用对体外培养的人牙髓干细胞（DPSCs）成牙分化的影响。方法：酶消化法获得DPSCs,采用碱性磷酸酶（ALP）活性检测和实时荧光定量RT—PCR测定牙本质涎磷蛋白1（DSPP）和牙本质基质蛋白1（DMP1）mRNA的表达以检测不同浓度人参皂苷Rgt（0．1,0．5,2．5,5,10,20μmol／L）及rhBMP-2（25,50,100,200rig／ha）单独及联合应用对DPSCs成牙分化的影响。结果：人参皂苷Rg1（2．5,5和10μmol／L）组的ALP活性明显高于同期对照组（P〈0．001）,5μmol／L组ALP活性明显高于其它浓度组（P〈0．001）;浓度为50,100,200mg/ml的rhBMP-2其ALP活性明显高于同期对照组（P〈0．001）,100ng／ml rhBMP-2组ALP活性最强。联合应用人参皂苷Rg1（5μmol／L）和rhBMP-2（100ng／ml）DSPP和DMP1 mRNA的表达明显高于单独应用组（P〈0．001）。结论：人参皂苷Rg1可促进体外培养的DPSCs成牙本质分化,人参皂苷：Rg1协同rhBMP-2能明显增强DISCs成牙本质分化。     

重组人骨形态发生蛋白9和2体外诱导成骨的活性分析

利用PCR扩增hBMP-9全长序列构建真核表达载体pcDNA4／HisMax—BMP-9,与质粒pSV2-dhfr共转染CHO—dhfr-细胞,以含有100μg／ml博来霉素的1MDM进行选择性培养,筛选抗性克隆,并用MTX扩增,提高rhBMP-9的表达量。经过细胞分离培养,得到稳定表达rhBMP-9的6株单克隆细胞株,ELISA法检测rhBMP-9表达水平,最高可达1．13μg/24h／10^6cells。收集最高表达蛋白的单克隆株培养基上清,经His标签的蛋白纯化柱纯化,冻干法浓缩得到100μm/ml的rhBMP-9。用浓度100μg／ml的rhBMP-9和rhBMP-2分别刺激培养C3H10,各项成骨指标显示rhBMP-9具有-定的体外诱导成骨能力,但不及rhBMP-2。     

Ras／Raf／MEK／Erk信号传导通路与低强度激光生物学效应

现代细胞生物学研究发现 ,蛋白质的磷酸化与去磷酸化作为传递各种信息的重要机制 ,在细胞生物学活动中起重要作用。蛋白激酶催化磷酸基团与蛋白质内特异的氨基酸共价结合 ,一系列的蛋白激酶及其磷酸化活化构成了信号传导级联反应〔1、2、3〕。丝裂素活化蛋白激酶 (ｍｉｔｏｇｅｎＡｃｔｉｖａｔｅｄｐｒｏｔｅｉｎｋｉ ｎａｓｅ ,ＭＡＰＫ)级联信号传导通路在信号传导过程中占据相当重要的地位 ,被认为是细胞外信号引起细胞增殖、分化等核反应的共同途径或汇聚点〔4、5〕。Ｒａｓ/Ｒａｆ/ＭＥＫ/Ｅｒｋ是ＭＡＰＫ级联途径中研究最为活跃也是…     

纳米羟基磷灰石/聚酰胺复合材料的人工肱骨头体内成骨中BMP-2及其基因表达研究

目的：对新型纳米晶骨修复和前建复合材料（n-HA／PA66）人工肱骨头的成骨能力及将其作为人工关节替代品的可行性进行评价。方法：新西兰大白兔20只，行双侧肱骨头切除，以nHA／PA人工肱骨头进行双侧肱骨头置换术。术后1、2、3、4、6、12、24周处死周分别处死动物。对假体与受体骨界面先行大体观察，然后分别用甲基丙烯酸甲酯（PMMA）制作硬组织片，甲苯胺兰染色和Masson染色后，对n-HA／PA人工肱骨头与受体骨界面进行BMP-2免疫组织化学检测和原位杂交BMP-2基因表达检测。结果：人工肱骨头在动物体内成骨的BMP-2及其基因表达研究显示，假体植入第3周，免疫组织化学染色显示BMP-2即有弱阳性表达，至6，12周为强阳性；第24周，BMP表达阳性率及阳性程度均有所降低。此外，原位杂交观察假体植入第1、2周，成骨细胞中BMP-2 mRNA表达呈弱阳性，第3周成骨细胞中BMP-2 mRNA表达呈强阳性，而在第4周，成骨细胞中表达阳性率和阳性程度均有所下降。研究显示人工肱骨头体内成骨的BMP-2及其基因表达高峰时间较文献报道的骨折模型晚。结论：n-HA／PA66人工肱骨头具有良好的成骨能力，作为新型人工关节材料为可与受体骨发生生物键合。     

呼吸道合胞病毒M2-1基因转染人肺腺癌细胞(PAa)后的电镜观察

目的：探讨呼吸道合胞病毒(RSV)M2-1基因致癌的超微形态学依据。方法：应用超薄切片电镜技术时比观察RSV M2-1基因转染前后人肺腺癌细胞(PAa)的超微结构改变。结果：转染后肺腺癌细胞(PAa／M2-1)呈现低分化超微结构特征：细胞膜表面微绒毛减少或缺如;细胞核明显增大,核浆比例增大,多见畸形核和双核,并可见巨大或多个核仁。核膜不规则,呈锯齿状或佛手状,核质富含常染色质。癌细胞胞浆少,以游离核糖体为主,其他细胞器稀少。偶见癌细胞凋亡。未转染肺腺癌细胞(PAa)呈现高分化超微结构特征：细胞表面有许多微绒毛;胞浆内有较多发育完好的细胞器。结论：RSVM2-1基因的转染和表达可诱导和促进PAa细胞去分化,其变化的分子机理有待进一步探讨。     

缺氧诱导因子1α对大鼠骨髓间充质干细胞核心结合因子α1表达的影响

目的：研究体外低氧实验中缺氧诱导因子1α（HIF-1α）对大鼠骨髓间充质干细胞核心结合因子α1（Cbfα1）表达的影响。方法：将体外培养的大鼠骨髓间充质干细胞分别置于常氧（含有5％c02的培养箱中）和低氧（含4％02、5％CO2和91％N2的三气培养箱中）中培养,分别于1d、3d、5d和7d用实时荧光定量PCR检测细胞内HIF-1α和CbfalmRNA的表达水平：用siRNA抑制细胞HIF-1αmRNA表达后用Westernblot法检测HIF-1α和Cbfal蛋白表达。结果：与常氧组相比,低氧组细胞HIF-1αanRNA的表达均增加（1d、3d、5d和7d：P〈0．05）,且3d达到最高峰;细胞CbfalmRNA的表达明显下降,3d尤为明显（P〈0．05）：低氧组细胞转染siRNA干扰HIF-1α表达后,能促进细胞内Cbfcd蛋白的表达（P〈0．05）。结论：低氧微环境抑制骨髓间充质千细胞的成骨向分化,细胞内HIF-1α反向调节Cbfal的表达。     

鼠重组腺病毒介导的骨形成蛋白-9转染大鼠牙囊干细胞的实验研究

目的：探讨鼠重组腺病毒介导的骨形成蛋白9基因转染大鼠牙囊干细胞的可行性及其转染后的成骨作用,以获得可用于牙周骨组织再生工程的基因修饰的种子细胞。方法：取大鼠下颌骨,解剖显微镜下体外分离培养纯化鉴定牙囊干细胞,腺病毒介导的骨形成蛋白9基因转染第三代牙囊干细胞,并设立空白对照组,绿色荧光病毒组（GFP组）。通过观察细胞形态及生长曲线变化,荧光显微镜及RT—PCR检测转染后骨形成蛋白9基因mRNA的表达,碱性磷酸酶及钙茜素红染色测定转染后牙囊干细胞的成骨活性。结果：与未转染对照组比较,转染组细胞转染2周后形成钙化结节,停滞期延长,数量轻度下降,倍增时间延长。牙囊干细胞转染骨形成蛋白9基因后12h后即有荧光表达,转染3,6,9,12d后骨形成蛋白9mRNA均呈阳性表达且逐渐增强,未转染对照组呈阴性。转染组碱性磷酸酶活性随转染时间的延长呈升高趋势,ALP染色及茜素红钙结节染色为阳性：未转染对照组碱性磷酸酶染色及钙茜素红染色均呈弱阳性表达,转染组显著高于未转染组。结论：鼠重组腺病毒介导的RBMP-9基因可以成功地转染大鼠牙囊干细胞,转染后牙囊干细胞高表达骨形成蛋白9,且具有明显的成骨作用。     

Cs（7DJ）＋Cs（6S1／2）间的碰撞能量转移

脉冲激光双光子激发Cs（6S1／2）到Cs（7Dj）态,在样品池条件下,利用原子荧光光谱方法研究了Cs（7Dj）＋Cs（6S1／2）的碰撞能量转移过程。利用三能级模型的速率方程分析,在不同的Cs（6S1／2）密度下,通过对直接荧光和转移荧光的时间积分荧光强度测量,得到了7D5／2→7D3／2精细结构转移截面为σ=（2．9±0．7）×10^-14cm^2。而碰撞转移到7Dj以外的猝灭截面分别为（7．4±2．1）×10^-14cm^2（对J=5／2）和（6．6±1．8）×10^-14cm^2（对J=3／2）。结合已有的实验结果,得到7DJ转移到8P态的截面分别为（0．6±0．3）×10^-14cm^2（对J=5／2）与（0．8±0．4）×10^-14cm^2（对J=3／2）。7D态主要是通过碰撞能量合并的逆过程[即7Dj＋6S→5D＋6P）猝灭。     

On2推出Hantro 9170 HD视频解码器

On2 Technologies公司推出第九代硬件视频编解码器设计Hantro 9170,该设计支持分辨率达全高清晰（ful HD）（1080p）、帧速60fps的多种格式视频播放,包括MPEG-1、MPEG-2、MPEG-4、Sorenson Spark．H．263．H．264。VC-1和REALVIDEO 8／9／10,以及高达66百万像素（megapixel）的JPEG静态图像。     

5-杂氮-2’-脱氧胞苷调控UCHL1基因表达对人前列腺癌PC-3细胞增殖及凋亡的影响

目的：探讨5-杂氮-2’-脱氧胞苷（5-Aza—CdR）对人前列腺癌细胞（PC-3）株增殖和凋亡的影响及其可能机制。方法以2．5、5．0、10．0kenol／L的5-Aza—CAR作用于PC-3细胞48h后,采用流式细胞术（FCM）检测细胞凋亡率;逆转录聚合酶链式反应（tiT—PcR）检测UCHL1 mRNA表达;甲基化测序聚合酶链反应（BsP）检测UCHLlCpG岛的甲基化状态;蛋白质印迹法（Western Blot）检测UCHL1蛋白的表达。结果：5-Aza—CAR对PC-3细胞生长有抑制作用;与对照组相比细胞凋亡率明显增高（P〈0．01）;5-Aza—CdR处理细胞后UCHL1的启动子甲基化水平明显降低（P〈0．01）;UCHL1 mRNA表达水平显著上调（P〈0．01）;UCHL1蛋白表达水平上升（P〈0．01）。结论：5-Aza—CAR能诱导前列腺癌PC-3细胞株凋亡的作用,其机制可能是5-Aza—CdR能逆转PC-3细胞UCHLl启动子CpG岛的异常甲基化,诱导mRNA转录和蛋白的表达。     

Multifunctional hydrogels that promote osteogenic hMSC differentiation through stimulation and sequestering of BMP2

The extracellular environment controls many cellular activities thereby linking external material cues to internal cell function. By better understanding these processes, synthetic extracellular material niches can be tailored to present cells with highly regulated physical and/or chemical cues that promote or suppress selected cell functions. Here, poly(ethylene glycol) (PEG) hydrogels were functionalized with fluvastatin-releasing grafts and growth factor binding heparin domains to enable the dynamic exchange of information between the material and cells from the outside-in and inside-out (i.e., bidirectional signaling). By incorporating a fluvastatin-releasing graft and carefully controlling the dose and temporal release, materials were designed to promote bone morphogenic protein (BMP2) and alkaline phosphatase (ALP) production by human mesenchymal stem cells (hMSCs). When the release of fluvastatin was controlled to occur over 2 weeks, BMP2 and ALP production was increased 2.2-fold and 1.7-fold, respectively, at day 28 compared to hMSCs cultured in the absence of fluvastatin. By introducing a heparin functionality into the gel to sequester and localize the hMSC-produced BMP2, the osteogenic differentiation of hMSCs was further augmented over fluvastatin delivery alone. Osteopontin and core binding factor α1 gene expression was 6-fold and 4-fold greater for hMSCs exposed to fluvastatin in the presence of the heparin functionalities, respectively. These results demonstrate how multifunctional gels that interact with cells in a bidirectional manner can efficiently promote selected cell functions, such as the osteogenic differentiation of hMSCs.     

Cs(7DJ)+H2的反应与非反应碰撞转移截面的测定

利用激光泵浦-吸收技术,研究了在样品池中的Cs(7D_J)+H₂→CsH[X¹∑+(v″=0)]+H光化学反应过程。双光子激发Cs-H₂混合蒸气中Cs原子至7²D态,荧光中除有泵浦能级发生的直接荧光外,还包含由精细结构碰撞转移产生的敏化荧光,CsH分子是由7D原子与H₂间的三体碰撞反应产生的。利用780 nm激光测量了CsH X¹∑+(v″=0→v″=21)吸收带。△Ⅰ′和△Ⅰ″分别表示泵浦7D_3/2和7D_5/2时的吸收光强。解速率方程组,得到7D_3/2→7D_5/2和7D_5/2→7D_3/2精细结构转移截面分别为(1.3±0.3)×10^-14和(9.8±2.0)×10^-15cm²。从7D_J碰撞到7D以外态的截面分别为(4.0±1.0)×10^-15(对J=3/2)和(3.6+0.9)×10^-15cm²(对J=5/2)。Cs(7D_J)+H₂→CsH+H的反应截面分别是(1.4±0.5)×10^-16(J=3/2)和(1.1±0.4)×10^-16cm²(J=5/2),7D_3/2与H₂的反应活动...更多性大于7D_5/2。     

Coupling BCP Ceramics with Micro-Vibration Stimulation Field for Cascade Amplification from Immune Activation to Bone Regeneration

The osteoimmunology has revealed that immune system plays an important role in maintaining bone metabolism and remodeling. As long-term physiological factor in bone, mechanical stimulation such as micro-vibration stimulation (MVS) exerts effects on regulating osteogenesis and immune response. In this study, the osteo-immunodulatory effects of bicalcium phosphate (BCP) ceramics coupled with MVS are investigated. This results find that the combination of BCP ceramics and MVS may exert synergistic effects on the polarization and functional status of macrophages through activating plasma membrance Ca²⁺ ATPase (PMCA) channel, reducing the intracellular calcium ion concentration, and inhibiting downstream extracellular signal-regulated kinase (ERK)1/2 signaling pathway. BCP ceramics coupled MVS could drive the macrophage polarization to wound-healing M2 phenotype to decrease the production of pro-inflammatory factors, enhance the secretion of anti-inflammatory cytokines and growth factors such as transforming growth factor (TGF)-β1 and bone morphogenetic protein (BMP)-2. Moreover, BCP and MVS-modulated macrophage secretion pattern can trigger the BMP/TGF-Smad signaling pathways to induce osteoblastic differentiation of bone marrow mesenchymal stromal cells (BM-MSCs) in vitro, and maintain cellular viability and promote the formation of collagen-rich osteoid like tissues and mature blood vessels in vivo. This study demonstrates that the introduction of mechanical stimuli like non-invasive MVS is an effective strategy to improve bone repair effects of biomaterials through endowing them with superior osteo-immunodulatory capacity.     

A Novel Approach to Enhance Bone Regeneration by Controlling the Polarity of GaN/AlGaN Heterostructures

Rapid and effective bone regeneration is still a major challenge in the clinical treatment of various bone diseases. Although recently developed electroactive materials have demonstrated high bone regeneration potential, the instability of the electrical stimulation and the unclear effects of the charge polarity on osteogenic differentiation hinder their clinical applications. In this work, GaN/AlGaN materials with well-controlled polarity are used for the first time to induce endogenous electric stimulation and facilitate bone regeneration. By controlling the direction and magnitude of the piezoelectric and spontaneous polarization in the functional layer (GaN), charged GaN/AlGaN surfaces of opposite polarity, whose zeta potentials are within the range of the physiological potential, are obtained. Compared with N-polarity GaN/AlGaN (with a positively charged surface), Ga-polarity GaN/AlGaN (with a negatively charged surface) nanofilms show rapid and superior bone repair in vivo. In addition, the Ga-polarity GaN/AlGaN hetero-structures significantly promote the attachment, spreading, recruitment, and osteogenic differentiation of bone mesenchymal stem cells in vitro. Moreover, the bone morphogenetic protein-6 (BMP6) expression profile in the early stages of osteogenic differentiation reveals that BMP6 may be an electrically sensitive osteogenic protein. This work sheds light on the application of III-nitride materials in bone regeneration.     

An NIR‐II Fluorescence/Dual Bioluminescence Multiplexed Imaging for In Vivo Visualizing the Location,Survival, and Differentiation of Transplanted Stem Cells

The in vivo distribution, viability, and differentiation capability of transplanted stem cells are vital for the therapeutic efficacy of stem cell–based therapy. Herein, an NIR‐II fluorescence/dual bioluminescence multiplexed imaging method covering the visible and the second near‐infrared window from 400 to 1700 nm is successfully developed for in vivo monitoring the location, survival, and osteogenic differentiation of transplanted human mesenchymal stem cells (hMSCs) in a calvarial defect mouse model. The exogenous Ag₂S quantum dot–based fluorescence imaging in the second near‐infrared window is applied for visualizing the long‐term biodistribution of transplanted hMSCs. Endogenous red firefly luciferase (RFLuc)‐based bioluminescence imaging (BLI) and the collagen type 1 promoter–driven Gaussia luciferase (GLuc)‐based BLI are employed to report the survival and osteogenic differentiation statuses of the transplanted hMSCs. Meanwhile, by integrating the three imaging channels, multiple dynamic biological behaviors of transplanted hMSCs and the promotion effects of immunosuppression and the bone morphogenetic protein 2 on the survival and osteogenic differentiation of transplanted hMSCs are directly observed. The novel multiplexed imaging method can greatly expand the capability for multifunctional analysis of the fates and therapeutic capabilities of the transplanted stem cells, and aid in the improvement of stem cell–based regeneration therapies and their clinical translation.     

Cover Picture: Biomineralized Polysaccharide Capsules for Encapsulation,Organization, and Delivery of Human Cell Types and Growth Factors (Adv. Funct. Mater. 6/2005)

The cover shows biomineralized polysaccharide capsules with specifiable make‐up, which can provide microenvironments for stabilization, growth, and differentiation of human cell types, as reported by Oreffo and co‐workers on p. 917. The capsules are amenable to complexation with a range of bioactive molecules and cells, offering tremendous potential as multifunctional scaffolds and delivery vehicles in tissue regeneration of hard and soft tissues. The construction of biomimetic microenvironments with specific chemical and physical cues for the organization and modulation of a variety of cell populations is of key importance in tissue engineering. We show that a range of human cell types, including promyoblasts, chondrocytes, adipocytes, adenovirally transduced osteoprogenitors, immunoselected mesenchymal stem cells, and the osteogenic factor, rhBMP‐2 (BMP: bone morphogenic protein), can be successfully encapsulated within mineralized polysaccharide capsules without loss of function in vivo. By controlling the extent of mineralization within the alginate/chitosan shell membrane, degradation of the shell wall and release of cells or rhBMP‐2 into the surrounding medium can be regulated. In addition, we describe for the first time the ability to generate bead‐in‐bead capsules consisting of spatially separated cell populations and temporally separated biomolecule release, entrapped within alginate/chitosan shells of variable thickness, mineralization, and stability. Such materials offer significant potential as multifunctional scaffolds and delivery vehicles in tissue regeneration of hard and soft tissues.     

NKG2D配体在视网膜母细胞瘤RB细胞的表达及意义

目的：探讨视网膜母细胞瘤RB细胞株上NKG2D配体的表达情况及其对同种异体NK细胞杀伤活性的影响。方法：采用半定量RT-PCR法检测RB细胞及人红白血病细胞K562细胞上NKG2D配体MICA、MICB、ULBP1、ULBP2、ULBP3的mRNA表达情况。应用MTT法检测人NK细胞对肿瘤细胞的杀伤活性。结果：K562细胞表面表达MICA、MICB、ULBP1、ULBP2、ULBP3,RB细胞表面表达MICA、MICB、ULBP2、ULBP3不表达ULBP1。K562细胞表面NKG2D的配体mRNA表达强度明显强于RB细胞（P〈0．05）。当效靶比分别为5：1、10：1、20：1、40：1时NK细胞对K562、RB细胞的杀伤活性分别为28．32％±2．06％、9．11％±0．89％;45．62％±1．17％、22．87％±1．38％;60．79％±1．78％、32．69％±0．37％;73．83％±3．05％、50．29％±2．49％,在各效靶比时NK细胞对K562细胞的杀伤活性较RB细胞明显增强（P〈0．05）。结论：NKG2D配体影响NK细胞对靶细胞的杀伤活性,NKG2D配体表达水平可能决定着NK细胞抗肿瘤免疫应答的强弱,提高NKG2D配体的表达有可能提高NK细胞的抗肿瘤作用。     

0D/1D Heterojunction Implant with Electro-Mechanobiological Coupling Cues Promotes Osteogenesis

Mimicking the natural bone extracellular matrix containing intrinsic topography and electrical signals is an effective way to modulate bone regeneration. However, simultaneously coupling of the intrinsic mechanobiology and electrical cues of implant to modulate bone regeneration remains ignored. Here, the authors report in situ designation of titanium dioxide (TiO₂) nanocone/bismuth oxide (Bi₂O₃) nanodot heterojunctions on bone implant surface to electro-biomechanically trigger osseointegration at bone/implant interface. TiO₂ nanocone/Bi₂O₃ nanodot heterojunctions exhibit built-in electric field at the nanoscale interface and elastic modulus equivalent to that of bone tissue. The nano-heterojunctions significantly promoted the attachment, spreading, and osteogenic differentiation of bone marrow mesenchymal stem cells in vitro, and the osteogenesis in vivo. The authors also show that the effects of nano-heterojunctions on osteogenesis are mediated by yes-associated protein biomechanical signal pathway and intracellular enrichment induced Phosphatidylinositol 3-kinase signal pathway. Their findings highlight the coupling of topographical and electric parameters of biomaterials for modulating cell behaviors.