美国国立卫生，AR53579，成骨：骨北回归线肽程增强 美国国立卫生研究院，AR056422，成骨肽合成代谢作用 美国国立卫生研究院，肌肉骨骼组织修复和再生，T32 AR050960 国防部，PC051219核仁：一种新型前列腺癌和骨髓内皮细胞粘附的调停 国防部，PC061508，前列腺癌转移细胞骨骨髓黏附调解

1。Biopanning 体内分离出噬菌体目标的长骨噬菌体DNA测序，并与相应的序列合成肽。肽合成甘氨酸 - 甘氨酸 - 甘氨酸，丝氨酸，赖氨酸连接器（链接器​​所需要的夫妇，帮助跟踪/追踪肽的荧光生色）。 <ol><li> 10周龄BALB / c小鼠被麻醉，暴露心脏。 </li><li>博士- 12噬菌体展示肽库试剂盒（New England Biolabs公司，富康，MA）是用于在体内biopanning，10 × 1010 PFU，并注入心脏。 </li><li>大约5分钟后，鼠标安乐死。暴露和解剖股骨，股骨的两端被切断，和骨髓被删除。 </li><li>左股骨内，外，用PBS冲洗10次，股骨添加到ER2537细菌（简称非洗脱噬菌体，请跳到步骤6）。 </li><li>右股骨交锁髓内运河的21号针头的2％干脱脂牛奶注射器冲洗10次，以去除非约束噬菌体。束缚噬菌体洗脱与盐酸甘氨酸缓冲液pH值2.2，瓦解1M三髓内运河，ER2537细菌，培养，扩增噬菌体（简称为洗脱）为4.5小时。 </li><li>细菌被拆除离心沉淀为10 ° C，而噬菌体沉淀在4 ° C过夜PEG / NaCl溶液中的另外4分钟。噬菌体是离心收集悬浮和沉淀两次，另外的1 / 6量的PEG /氯化钠。最后沉淀悬浮在TBS的0.02％叠氮化钠。 </li><li>洗脱和非洗脱噬菌体扩增洗脱液分别注入不同的小鼠，上述隔离和噬菌体。只有从注射非洗脱噬菌体，股骨直接放入细菌（见上述第4步）鼠标左股骨。洗脱噬菌体鼠标的分离只能从右侧股骨和盐酸甘氨酸缓冲液（见上面的步骤5）前公布增加细菌的噬菌体。 </li><li> 在体内 biopanning程序重复使用- 98底漆（噬菌体展示套件提供）的DNA测序前的三倍。 </li><li>汇集噬菌体在细菌培养中扩增，纯化，定量分析，然后分别注入了第四鼠标。序列是重复。 </li><li>为了展示肽特异性骨和骨髓，噬菌体洗脱从肾脏和肝脏进行了分析如上所述。在全部四轮比赛中有少量分布在这些器官（小于10％）的噬菌体。 </li><li>合成肽与甘氨酸-甘氨酸-甘氨酸，丝氨酸，赖氨酸序列，与无素细胞和组织染色，以及在体内工作。 </li></ol> 2。间质细胞培养成骨细胞分化实验<ol><li>克隆小鼠多能干骨髓基质（D1）在我们的实验室1中分离的细胞系在体外实验中使用。细胞培养贝科必要的修改培养基，DMEM培养液，低血糖（GIBCO猫＃11885-084），辅以10％小牛血清（GIBCO猫＃16000-044），50毫克每毫升抗坏血酸钠（西格玛猫＃A7631 ），100 U / mL青霉素G和100毫克/毫升链霉素（GIBCO猫＃15140-122）。 </li><li>文化均保持在37 ° C直到在饱和湿度5％的CO 2水套式孵化器和子培养细胞约90％汇合。 </li><li>细胞胰酶消化，离心来沉淀，计数，并在5X10 3细胞/厘米2组织培养处理的塑料制品的浓度接种。 </li></ol> 3。肽染色培养细胞和组织<ol><li> D1或骨髓细胞接种于纤维连接蛋白或明胶涂层室以及幻灯片的DMEM和10％的胎儿24小时的牛血清。 </li><li>用PBS洗涤细胞。 30分钟，用4％多聚甲醛固定细胞/组织</li><li>过量的醛被封锁10分钟使用0.5 M甘氨酸（pH值7.5）。 </li><li> 5％BSA +亲和阻塞的解决方案除了阻止细胞表面的受体的内源性抗生物素蛋白和生物素是由1个小时，然后用5％BSA +生物素封闭液封闭1小时5分钟，PBS洗涤一次。 </li><li> 100微米生物素L7和R1的肽分别加入30分钟井。 </li><li>细胞分别用PBS冲洗5分钟，每个1:1000稀释Alexa的面粉链霉和在黑暗中孵育30分钟三次。 </li><li>洗涤细胞和盖玻片vectashield安装媒体安装，并在荧光显微镜下观察。 </li></ol> 4。碱性磷酸酶活性测定<ol><li>碱性磷酸酶彗星使用碱性磷酸酶试剂盒（BioRad公司）制造商的建议的单层olorimetric检测。 </li><li> D1的细胞接种在5 × 10 3细胞/ cm 2的 6孔培养板的密度，并与R1肽治疗，ALP活性进行了量化，在4天，8，12，16和20。 </li><li>要准备的细胞裂解液，用PBS缓冲液洗细胞层三次，然后到PBS刮其次是超声和离心去除细胞碎片。 </li><li>然后混合等量的裂解液（100μL）与100μL新鲜配制的色度基板段，硝基苯磷酸盐，孵育在37 ° C为30分钟。 </li><li> 100μL的0.2 N NaOH溶液中加入酶反应停止。 ALP活性测定在405 nm处用酶标仪（BIO - RAD）。 </li><li>用BioRad公司蛋白定量试剂盒，细胞裂解液中蛋白质含量测定AP的活动，然后表示nmol /分钟的/毫克蛋白质产生的第 - 硝基酚。 </li><li>根据制造商的规格（Sigma公司，公司）ALP活性。 </li><li> ALP活性单位正常化毫克总蛋白含量与BCA法测定细胞裂解液（皮尔斯，罗克福德，IL）。 </li></ol> 5。使用L7和R1的间质细胞分化<ol><li> Subconfluent间质细胞（D1），增长约90％汇合，5NM L7和R1的肽治疗。 </li><li> 0.5，2，6，24和48小时，收获细胞的RNA采用Qiagen公司的RNeasy试剂​​盒，使用制造商的指示。 </li><li>逆转录酶（RT）反应退火（37℃，10分钟）和第一链cDNA合成（42℃，30分钟），热灭活（95℃5分钟）。由此产生的基因储存冷冻（-70℃），直到通过实时PCR检测。 </li><li>实时PCR使用的QuantiTect SYBR绿色PCR试剂盒（QIAGEN）。反应进行25μLSYBR绿色套件预混，正向和反向引物如Runx2的，Osterix，骨钙素，骨涎蛋白，Wnt信号通路的基因，如b - catenin的骨特异性基因（300 nmol / L的）， LRP6，Wnt信号5A，7B，10B，DKK1和OPG和RANKL（见表1）。 </li><li> cDNA样本3μL添加到RT反应未稀释的最终反应混合物。 96实时PCR格式包括六个重复的质粒DNA标准的10倍稀释。板井密封光学胶盖（BioRad公司），在低速离心（300克，5分钟），以确保完全混合。 </li><li>每个样品分析，至少一式两份，每份用iCycler仪（BioRad公司实验室，大力士，CA）。 </li><li> PCR使用的协议涉及AmpliTaq黄金DNA聚合酶的激活，40个循环，随后在94 ° C为30秒，在72 ° C 30秒30秒，并扩展各自的退火温度。 PCR循环阈值，每个样品扫描（CT）计算荧光超过阈值限制的地步。沿着线性对数生长期在10到20个以上标准偏差（SDS）的平均背景荧光的荧光曲线的门槛限制是固定的。标准曲线方程计算回归分析与LOG10的cDNA相对于总RNA目前的平均CT。骨特异性基因的靶基因的相对表达归18S和B -肌动蛋白是Wnt信号通路的基因。 </li></ol> 在体内制备的明胶海绵复合材料6 。 <ol><li>使用无菌例程，明胶海绵缸（法玛西亚普强猫＃09-0353-01），8毫米长，直径3毫米，使用自行设计的仪器。 </li><li>手术前二十四小时，明胶海绵气瓶浸泡装有L7或R1肽（PBS中20微米）或PBS缓冲液无肽。明胶海绵气瓶被转移到无菌的12孔培养皿中，存放在-4 ° C过夜</li><li>在手术时，在培养皿中的明胶海绵复合搬出冰箱和雪藏，直到手术。 </li></ol> 7。骨缺损<ol><li>卫生系统在弗吉尼亚大学的动物护理和使用委员会前，正在执行的所有动物的程序获得批准。十人九月龄成年同源男性菲舍尔344大鼠（350-400克），氯胺酮（50克）每克体重和甲苯噻嗪（5克，每克体重）的混合腹腔麻醉。 </li><li>一个双边的手术方法是胫骨内侧方面。在这岁或以上的大鼠显示微不足道的横截面积增加，皮质骨面积，骨量，骨密度，轴向长度的anteromedia升方面的胫骨。测量3毫米宽8毫米长期使用气动毛刺（馆高速钻;吉玛）生理盐水冲洗下，创建一个大型的，长方形的unicortical缺陷。 </li><li>治疗骨缺损是用明胶海绵或不肽。所有植入支架，3毫米宽，8毫米的长度由1.5毫米的深度测量插入温柔攻。任何内部或外部肢体固定装置术后使用，并允许不受限制地走动的动物后立即手术。 </li></ol> 8。组织学和形态计量学大体形态和所采取的数字图像的骨再生进行了评估。 <ol><li>安乐死大鼠的胫骨，3，5和12周进行量化损伤反应中产生的新骨量。 </li><li>收获胫骨缺损仔细检查后，被固定在10％中性缓冲福尔马林，RDO快速decalcifier脱钙72小时，然后常规处理和石蜡包埋，并分段纵向。 </li><li>骨再生进行了评估总值和光镜检查。 </li><li>十大动物被用于每个条件（即空的缺陷，明胶海绵单独和明胶海绵加R1的肽）。代表跨〜40的组织切片，每个8微米厚的8mm unicortical缺陷。的40部分，我们使用了至少6个部分，以量化的新骨量。 </li><li>所有的组织切片染色与苏木/伊红（H＆E），标签骨样基质。 </li><li>在显微镜下，所有的幻灯片进行了评估。 </li></ol> 9。骨修复概述骨修复显然是肌肉骨骼组织再生领域的一个非常重要的考虑的领域。肽目标骨矫形外科实践中有显着的实用，特别是在使用多年，如果不是几十年，可以留置的protheses。仿生骨热带因素的属性，可以创建在组织工程领域的技术向前迈进一步，以及为骨修复手术。针对合成或天然聚合物因素的骨内可协助特异性细胞黏附，从而清除内源性细胞进行修复和再生的组织。另外一个目标是要建立，如果我们独特的骨针对biopanning体内噬菌体展示库中确定由肽将绑定到骨骼和骨髓细胞，并有调节成骨在体外和体内骨再生的潜力。 在我们的实验中，我们交付使用天然和合成聚合物基质和骨再生骨再生的组织学，生物化学，基因表达，显微CT，以生物力学的研究，以阐明肽活动的细节的大鼠股骨缺损肽。这种方法的进一步发展可导致目标的骨生物活性化合物的发现和发展，使可能通过修复机制，在细胞和分子水平生物学特征。 染色与生物素标签，以确定具有约束力的和本地化的间质细胞合成肽。异硫氰酸荧光素（FITC）标记的抗生物素蛋白是用于本地化显微镜的肽在体外细胞生长的约束力。肽结合文化中的间质细胞，以及骨骼和骨髓在体外鼠标肋骨组织切片（ 图 1 ）。 Von Kossa染色的一间质细胞（D1），是从骨髓中克隆和确定的肽在体外的成骨细胞的影响。细胞治疗与肽L7和R1，以确定在细胞形态和基因表达的变化。在第4天，细胞开始聚集，并随着时间的推移，形成结节，一个细胞的行为模式相似，在培养骨细胞（ 图2）扩大的聚合。 此外L7的肽或R1加强与冯科萨的文化细胞染色。细胞，然后用不同浓度高达16天中文化L7的肽或R1。经测定，5纳米肽在体外是最有效的。在对待文化与肽的碱性磷酸酶活性增加2倍和3.5倍之间。倍的增长，取决于肽的浓度和文化的治疗时间。 实时PCR 与L7和R1肽处理的细胞进行了分析，使用实时P训练班两个已知的成骨细胞转录因子（Osterix和Runx2的）和骨基质蛋白（骨涎蛋白，骨钙素和I型胶原）的基因表达。治疗与R1显示Osterix和Runx2的基因表达增加。型胶原的基因表达维持不变。与L7的处理表明了BSP和骨钙素基因表达的增加以及I型胶原蛋白基因的表达。这表明，这两个肽间质细胞培养成骨的发生和进展有关的基因的效果。 在体外细胞培养与肽R1和L7的治疗揭示了一个相关的骨形成间质细胞的合成代谢作用。护骨素以RANKL的比例在增加，显示在招聘骨细胞作为破骨细胞的骨吸收减少。肽细胞的治疗，也导致在与骨形成和减少的因素，抑制成骨基因的表达的增加。 组织学要确定的骨量，我们暴露的骨片μLtraviolet光，从而产生自体荧光。 2定量的目的，这提供了更大的的骨骼和非骨组织之间的对比。被拍到的部分使用尼康数码成像系统，在同一放大倍数（X10客观）明场和紫外灯（ 图3）尼康和紫外线。 Metavue成像软件（Molecular Devices公司）所产生的数字图像进行分析。我们选择了一个固定的，利益的矩形区域（ROI），包含8毫米unicortical缺陷。损伤部位始终代表手动放置在每个图像上的正确位置的框，这里面投资回报率。 H＆E染色阳性像素部分自动使用魔术棒工具，设置一种颜色宽容。这种宽容正是在H＆E -染色切片组织学外观与骨组织的绿色范围内，对应的突出像素的设置。每个部分的H＆E -积极像素总数录得。个别路段的像素数，平均每个胫骨样本，并在各治疗组之间的差异，基于这些平均数计算。所有样品的地区，然后进行比较的基础上的新皮质骨量（ 图4）。 骨中存在的部分进行了量化，以确定在单独比较，明胶海绵用明胶海绵+肽治疗骨缺损的修复。填充明胶海绵+ R1的肽的缺陷显示10金额最大的皮质修复，7和2倍，3，5，12周分别。明胶海绵的差异，在皮质骨修复+肽相比，单用明胶海绵在3和5周最为显着，表明该肽促进骨皮质的再生（图5）。 10。成骨肽的合成代谢作用 OPG / RANKL的比例大于RANKL / OPG的比例，这表明，肽对成骨细胞有直接的影响和调节骨吸收。 合成肽可能有较大的分子，如速度的准备，分子稳定，保质期长和潜在的治疗应用，的优势。试图调节骨质流失，并通过控制细胞因子和生长因子的再生，已经取得了重大进展。这些osteotropic肽，以现有的疗法，刺激骨的合成代谢和骨再生提供有吸引力的替代品。 <img src="/files/ftp_upload/2362/2362fig1.jpg" alt="图1" /> 图1。鼠标罗纹组织在体外的部分骨髓和骨肽结合。 <img src="/files/ftp_upload/2362/2362fig2.jpg" alt="图2" /> 图2（D1），间质细胞形成肽治疗后结节。 <img src="/files/ftp_upload/2362/2362fig3.jpg" alt="图3" /> 图3。使用成骨肽，L7和R1的骨修复的组织学。 <img src="/files/ftp_upload/2362/2362fig4.jpg" alt="图4" /> 图4。骨部分与H＆E染色，紫外光照射下，这会导致自体荧光。这提供了更大的骨骼和非骨组织之间的对比定量分析的目的。 <img src="/files/ftp_upload/2362/2362fig5.jpg" alt="图5" /> 图5。定量组织学部分骨皮质修复的金额最大的5个星期10倍的变化，7和3的2倍和5周表明，该肽促进骨皮质再生。

<ol>
	<li>Diduch, D. R., Coe, M. R., Joyner, C., Owen, M. E., Balian, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+cell+lines+from+bone+marrow+that+differ+in+terms+of+collagen+synthesis,+osteogenic+characteristics,+and+matrix+mineralization.">Two cell lines from bone marrow that differ in terms of collagen synthesis, osteogenic characteristics, and matrix mineralization.</a> J. Bone Joint Surg. Am. 75, 92-105 (1993).</li><li>Stoddart, M. J., Furlong, P. I., Simpson, A. E., Davies, C. M., Richards, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viability+in+ex+vivo+cultured+cancellous.">Viability in ex vivo cultured cancellous.</a> Eur. Cells. Mater. 13, 69-70 (2007).</li><li>Lehr, J. E., Pienta, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preferential+adhesion+of+prostate+cancer+cells+to+a+human+bone+marrow+endothelial+cell+line.">Preferential adhesion of prostate cancer cells to a human bone marrow endothelial cell line.</a> J. Natl. Cancer Inst. 90, 118-123 (1998).</li><li>Schuster, N., G&#246;tz, C., Faust, M., Schneider, E., Prowald, A., Jungbluth, A., Montenarh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wild-type+p53+inhibits+protein+kinase+CK2+activity.">Wild-type p53 inhibits protein kinase CK2 activity.</a> J. Cell Biochem. 81, 172-183 (2001).</li><li>Cwirla, S. E., Peters, E. A., Barrett, R. W., Dower, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptides+on+phage:+a+vast+library+of+peptides+for+identifying+ligands.">Peptides on phage: a vast library of peptides for identifying ligands.</a> Proc. Natl. Acad. Sci. U. S. A. 87, 6378-6382 (1990).</li><li>Doorbar, J., Winter, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+a+peptide+antagonist+to+the+thrombin+receptor+using+phage+display.">Isolation of a peptide antagonist to the thrombin receptor using phage display.</a> J. Mol. Biol. 244, 361-369 (1994).</li><li>Ellerby, H. M., Arap, W., Ellerby, L. M., Kain, R., Andrusiak, R., Rio, G. D., Krajewski, S., Lombardo, C. R., Rao, R., Ruoslahti, E., Bredesen, D. E., Pasqualini, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-cancer+activity+of+targeted+pro-apoptotic+peptides.">Anti-cancer activity of targeted pro-apoptotic peptides.</a> Nat. Med. 5, 1032-1038 (1999).</li><li>Goodson, R. J., Doyle, M. V., Kaufman, S. E., Rosenberg, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-affinity+urokinase+receptor+antagonists+identified+with+bacteriophage+peptide+display.">High-affinity urokinase receptor antagonists identified with bacteriophage peptide display.</a> Proc. Natl. Acad. Sci. U. S. A. 91, 7129-7133 (1994).</li><li>Pasqualini, R., Koivunen, E., Ruoslahti, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+peptide+isolated+from+phage+display+libraries+is+a+structural+and+functional+mimic+of+an+RGD-binding+site+on+integrins.">A peptide isolated from phage display libraries is a structural and functional mimic of an RGD-binding site on integrins.</a> J. Cell Biol. 130, 1189-1196 (1995).</li><li>Pasqualini, R., Koivunen, E., Ruoslahti, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha+v+integrins+as+receptors+for+tumor+targeting+by+circulating+ligands.">Alpha v integrins as receptors for tumor targeting by circulating ligands.</a> Nat. Biotechnol. 15, 542-546 (1997).</li><li>Pasqualini, R., Koivunen, E., Kain, R., Lahdenranta, J., Sakamoto, M., Stryhn, A., Ashmun, R. A., Shapiro, L. H., Arap, W., Ruoslahti, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aminopeptidase+N+is+a+receptor+for+tumor-homing+peptides+and+a+target+for+inhibiting+angiogenesis.">Aminopeptidase N is a receptor for tumor-homing peptides and a target for inhibiting angiogenesis.</a> Cancer Res. 60, 722-727 (2000).</li><li>Rajotte, D., Arap, W., Hagedorn, M., Koivunen, E., Pasqualini, R., Ruoslahti, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+heterogeneity+of+the+vascular+endothelium+revealed+by+in+vivo+phage+display.">Molecular heterogeneity of the vascular endothelium revealed by in vivo phage display.</a> J. Clin. Invest. 102, 430-437 (1998).</li><li>Rajotte, D., Ruoslahti, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+dipeptidase+is+the+receptor+for+a+lung-targeting+peptide+identified+by+in+vivo+phage+display.">Membrane dipeptidase is the receptor for a lung-targeting peptide identified by in vivo phage display.</a> J. Biol. Chem. 274, 11593-11598 (1999).</li><li>Wang, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+high-affinity+peptide+antagonists+of+14-3-3+proteins+by+phage+display.">Isolation of high-affinity peptide antagonists of 14-3-3 proteins by phage display.</a> Biochemistry. 38, 12499-12504 (1999).</li><li>Sugahara, K. N., Teesalu, T., Karmali, P. P., Kotamraju, V. R., Agemy, L., Greenwald, D. R., Ruoslahti, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coadministration+of+a+tumor-penetrating+peptide+enhances+the+efficacy+of+cancer+drugs.">Coadministration of a tumor-penetrating peptide enhances the efficacy of cancer drugs.</a> Science. 328, 1031-1035 (2010).</li><li>Sikes, R. A., Cooper, C. R., Beck, G. L., Pruitt, F., Brown, M. L., Balian, G., Meadows, G., Kluwer, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+Stromal+Cells+as+Therapeutics+Targets+in+Osseous+Metastasis.">Bone Stromal Cells as Therapeutics Targets in Osseous Metastasis.</a> Cancer Growth and Progression. "Integration/Interaction of Oncologic Growth". , 369-386 (2005).</li><li>Santos, J. L., Pandita, D., Rodrigues, J., P&#234;go, A. P., Granja, P. L., Tom&#225;s, H., Balian, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor-Mediated+Gene+Delivery+in+Mesenchymal+Stem+Cells+using+PAMAM+Dendrimers+conjugated+with+Osteotropic+Peptides.">Receptor-Mediated Gene Delivery in Mesenchymal Stem Cells using PAMAM Dendrimers conjugated with Osteotropic Peptides.</a> Molecular Pharmaceutics. , (2010).</li></ol>

生产此视频赞助New England Biolabs公司，公司，从而产生一些本文中使用的试剂。

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peptides from phage display library modulate gene expression in mesenchymal cells and potentiate osteogenesis in unicortical bone defects

两种新型合成肽加速骨形成和可交付使用的胶原蛋白基质。这项研究的目的是探讨在unicortical缺损模型骨修复的影响。治疗间质细胞产生碱性磷酸酶活性的增加，表明细胞结节的形成，并提高Runx2的，osterix，骨涎蛋白和骨钙素基因的表达。胶原蛋白海绵浸泡肽促进骨缺损的修复，而控制效果较差。这项研究的结果表明，对成骨肽在体外治疗的间质细胞分化，并且，在体内交付使用胶原海绵肽促进unicortical缺陷的修复。

Watch this Scientific Journal Video about Peptides from Phage Display Library Modulate Gene Expression in Mesenchymal Cells and Potentiate Osteogenesis in Unicortical Bone Defects at JoVE.com

Peptides from Phage Display Library Modulate Gene Expression in Mesenchymal Cells and Potentiate Osteogenesis in Unicortical Bone Defects

噬菌体展示库，用于识别目标的骨肽序列。其目的是调查这些肽的间质细胞分化的影响，以确定其对骨再生的效果。

从噬菌体抗体库的肽调节间质细胞的基因表达和使可能在Unicortical骨缺损的成骨

Two novel synthetic peptides accelerate bone formation and can be delivered using a collagen matrix. The aim of this study was to investigate the effects ...

peptides-from-phage-display-library-modulate-gene-expression

Research

JoVE Journal

Biology

17.9K 次观看.  University of Virginia. 噬菌体展示库，用于识别目标的骨肽序列。其目的是调查这些肽的间质细胞分化的影响，以确定其对骨再生的效果。

视频: Peptides from Phage Display Library Modulate Gene Expression in Mesenchymal Cells and Potentiate Osteogenesis in Unicortical Bone Defects

Culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. 

This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.

文化髓系树突状细胞从骨髓前体

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to ...

科研

生物学

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

主电穿孔的小鼠骨髓造血细胞培养制备

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell differentiation, axon guidance, retinotopic organization and synapse formation[1]. One drawback is the inability to efficiently and reliably manipulate gene expression in RGCs in vivo, especially in the otherwise accessible murine visual pathways. Transgenic mice can be used to manipulate gene expression, but this approach is often expensive, time consuming, and can produce unwanted side effects. In chick, in ovo electroporation is used to manipulate gene expression in RGCs for examining retina and RGC development. Although similar electroporation techniques have been developed in neonatal mouse pups[2], adult rats[3], and embryonic murine retinae in vitro[4], none of these strategies allow full characterization of RGC development and axon projections in vivo. To this end, we have developed two applications of electroporation, one in utero and the other ex vivo, to specifically target embryonic murine RGCs[5, 6]. 
With in utero retinal electroporation, we can misexpress or downregulate specific genes in RGCs and follow their axon projections through the visual pathways in vivo, allowing examination of guidance decisions at intermediate targets, such as the optic chiasm, or at target regions, such as the lateral geniculate nucleus. Perturbing gene expression in a subset of RGCs in an otherwise wild-type background facilitates an understanding of gene function throughout the retinal pathway. Additionally, we have developed a companion technique for analyzing RGC axon growth in vitro. We electroporate embryonic heads ex vivo, collect and incubate the whole retina, then prepare explants from these retinae several days later. Retinal explants can be used in a variety of in vitro assays in order to examine the response of electroporated RGC axons to guidance cues or other factors. In sum, this set of techniques enhances our ability to misexpress or downregulate genes in RGCs and should greatly aid studies examining RGC development and axon projections.

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties.

在子宫内和前体内的基因表达在小鼠视网膜神经节细胞的电穿孔

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

使用基因脉冲MXcell电穿孔系统主细胞的转染效率高

Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects

The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and functional tissue. Matrix associated stem cell implants (MASI) aim to regenerate cartilage defects due to arthritic or traumatic joint injuries. Adult mesenchymal stem cells (MSCs) have the ability to differentiate into cells of the chondrogenic lineage and have shown promising results for cell-based articular cartilage repair technologies. Autologous MSCs can be isolated from a variety of tissues, can be expanded in cell cultures without losing their differentiation potential, and have demonstrated chondrogenic differentiation in vitro and in vivo1, 2.
In order to provide local retention and viability of transplanted MSCs in cartilage defects, a scaffold is needed, which also supports subsequent differentiation and proliferation. The architecture of the scaffold guides tissue formation and permits the extracellular matrix, produced by the stem cells, to expand. Previous investigations have shown that a 2% agarose scaffold may support the development of stable hyaline cartilage and does not induce immune responses3.
Long term retention of transplanted stem cells in MASI is critical for cartilage regeneration. Labeling of MSCs with iron oxide nanoparticles allows for long-term in vivo tracking with non-invasive MR imaging techniques4.
This presentation will demonstrate techniques for labeling MSCs with iron oxide nanoparticles, the generation of cell-agarose constructs and implantation of these constructs into cartilage defects. The labeled constructs can be tracked non-invasively with MR-Imaging.

Goal of the presentation is to demonstrate a highly reproducible method to generate matrix associated stem cell implants in cartilage defects, which can be visualized with MR imaging. Stem cells are labeled with FDA-approved Ferumoxides, mixed with agarose, implanted into cartilage defects and imaged with a 7T MR scanner.

Ferumoxides植入人类间质干细胞在软骨缺损的标记

The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

使用自动细胞计数器简化基因表达研究：在Namalwa细胞的IL - 4依赖的基因表达的siRNA击倒

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

在体外重组活跃T。谷盗端粒酶

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions. The technique has largely been used to discover protein-protein interactions but the bait molecule to be challenged need not be restricted to proteins. The protocol presented here has been optimized to allow a modest number of baits to be screened in replicates to maximize the identification of independent clones presenting the same protein. This permits greater confidence that interacting proteins identified are legitimate interactors of the bait molecule. Monitoring the phage titer after each affinity selection round provides information on how the affinity selection is progressing as well as on the efficacy of negative controls. One means of titering the phage, and how and what to prepare in advance to allow this process to progress as efficiently as possible, is presented. Attributes of amplicons retrieved following isolation of independent plaque are highlighted that can be used to ascertain how well the affinity selection has progressed. Trouble shooting techniques to minimize false positives or to bypass persistently recovered phage are explained. Means of reducing viral contamination flare up are discussed.

Phage display is a powerful technique to capture proteins or protein moieties that interact with an immobilized molecule of interest. Once a decision of the type of phage cDNA library to create and screen has been made, the protocol described here permits efficient affinity selection leading to identification of interactors.

一个协议，用于噬菌体展示和亲和选择以重组蛋白诱饵

Using  recombinant phage as a scaffold to present various protein portions encoded by  a directionally cloned cDNA library to immobilized bait molecules ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

siRNA的筛选识别泛素和泛素样系统压力表生物学途径在培养的哺乳动物细胞

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.