Research was partly supported by NIH research grants R01 AR062173 and SHC 250862 to IEA. ES is the recipient of NIH T32 CTSC predoctoral fellowship.

中sRANKL MC DNA 1。水动力传递<ol><li>通过小鼠尾静脉流体力学交货<ol><li>尾静脉注射前称量鼠标。中的小鼠体重的10％〜的总体积稀释sRANKL或绿色荧光蛋白（GFP）的MC在林格氏溶液（预暖，在37℃）。 </li><li>热身鼠标在一个笼子10分钟前注射，以扩张血管，使侧脉（LVS）可见。监视鼠标小心，以避免脱水和高热。 </li><li>只要LV的被扩张，可见，鼠标转移到阻挡并将插头远远不够倒入桶内，以抑制运动。监视鼠标正常活动如呼吸。如果需要调整限制器的插头。 </li><li>从尾尖用酒精擦拭消毒注射区的3/4。使用27-30号针头的小鼠尾静脉注射。紧紧握住尾巴用一只手插入针到尾静脉。施加压力的注射器，并在5-7秒完成注射。 </li><li>从尾静脉中取出针头并用棉球将压力施加于注射部位止血。监视鼠标15-30分钟，再用鼠标转回动物饲养，一旦呼吸率降低到正常速度。 </li></ol></li><li>的小鼠血清TRACP5b的帖子基因转移系统性鼠标sRANKL的表达和定量确认<ol><li>热身鼠标在一个笼子里5-10分钟，以扩张血管，使的LV可见。监视鼠标小心，以避免脱水和高热。 </li><li>作一小切口用刀片将鼠标的尾部在一个站点1列从尾部的尖端。 </li><li>收集约100微升的血液中的血清分离管中。 </li><li>孵育血清分离管在室温下放置30分钟。 </li><li>以10,000×g离心5分钟，离心样品并收集血清。 STOR在-80°C，E无血清作进一步的分析。 </li><li>使用市售的试剂盒血清样本进行sRANKL ELISA和小鼠血清TRACP5b的检测。 </li></ol></li></ol> 2，体外破骨细胞生成的鼠标桥梁养护管理<ol><li>隔离和桥梁养护管理的培养<ol><li>胫骨和股骨骨隔离：安乐死的供体小鼠与CO 2，消毒腿用70％乙醇。从耻骨解剖到跟骨，以除去股骨和胫骨完好无损。 </li><li>小心地取出皮肤和肌肉不破坏骨。放置处理股骨和胫骨中含有磷酸盐缓冲盐水（PBS）的培养皿中。 </li><li>去除骨髓：切断对股骨和胫骨的骨骼的一侧的前端和冲洗出骨髓入50毫升的管中，使用1ml注射器用25号针头，装有含1％青霉素/链霉素的α-MEM （破骨细胞培养基/ OCM）。 </li><l我&gt;过程骨髓：混合并通过一个70微米尼龙细胞过滤器传递给它的骨髓悬浮液转移至新的50ml管中。离心将细胞以300×g离心并重新悬浮在α-MEM中含有1％青霉素/链霉素和10％胎牛血清（破骨细胞培养基/ OCM（+））。 </li></ol></li><li>巨噬细胞的培养<ol><li>使用血球或自动细胞计数器计数细胞。 </li><li>板1×10 6每孔细胞的6孔板或3×10 5细胞在玻璃盖玻片（5毫米）或牙本质片放置在96孔板中孵育在37℃下</li><li>吸出物含有非贴壁细胞和培养细胞在37℃下用含有25纳克/毫升鼠M-CSF 48小时新鲜OCM（+）的所有介质。 </li><li>含有25毫微克/毫升鼠标M-CSF和30毫微克/毫升小鼠sRANKL启动破骨细胞吸出所有媒体和培养细胞在37℃的新鲜OCM（+）的媒体。根据需要补充媒体每3天。 </li><li>细胞生长约6-8天以形成能够再吸收骨巨多核细胞。只要多核细胞出现，执行TRAP（抗酒石酸酸性磷酸酶），使用市售的试剂盒染色，而当完全成熟，进行功能分析。直观上用鬼笔环肽染色和图像附着用扫描电子显微镜（SEM）如先前19描述牙本质片的细胞的盖玻片的F-肌动蛋白环。 </li></ol></li></ol> 3， 在体外破骨细胞生成从人PBMC <ol><li>人外周血单个核细胞的分离<ol><li>加入10 ml预热的Histopaque-1077的入50毫升的试管中。 </li><li>从用无菌PBS血库获得到50ml试管（1:1比例的血用PBS）空白细胞过滤器。层中的稀释血液在HISTOPAQUE溶液非常缓慢，并确保血液不与HISTOPAQUE混合。 </li><li>在1000×g离心20分钟，离心样品在18°C。跟随ING离心，小心分离含有白细胞的白色血沉棕黄层和层转移到新的50ml管中。 </li><li>在650×g离心5分钟，再稀释细胞，用PBS离心以收集细胞。 </li><li>在OCM去除上清，重悬细胞（ - ）介质，并使用细胞计数器计数细胞。 </li></ol></li><li>电镀与外周血单个核细胞培养<ol><li>重悬的细胞在OCM（+）介质和板8×10 6个细胞/ ml，每孔中×10 6个细胞/ ml，每孔在96孔板的6孔板或1。根据需要为免疫荧光成像，以及对骨片适于骨吸收测定法作为在玻璃盖玻片（5毫米）的板的细胞先前所描述20。 </li><li>改变培养基，补充细胞与人M-CSF，每2-3天或按要求。然后通过添加人sRANKL的30纳克/毫升连同25毫微克/毫升的人M-CSF在OCM（+）媒体发起osteoclastogenes进行到第5天的分化步骤是。 </li><li>生长细胞为约14至21天，以形成巨型多核细胞。只要多核细胞出现，这些细胞可以被标记为TRAP，并且当完全成熟，功能分析可以被执行。 F-肌动蛋白环可以可视化使用鬼笔环肽染色盖玻片。细胞的形态，可以检查连接在用SEM对牙本质切片的细胞​​。 </li></ol></li></ol>

<ol>
	<li>Yelin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cost+of+musculoskeletal+diseases:+impact+of+work+disability+and+functional+decline.">Cost of musculoskeletal diseases: impact of work disability and functional decline.</a> The Journal of rheumatology. Supplement. 68, 8-11 (2003).</li><li>Boyce, B. F., Rosenberg, E., de Papp, A. E., Duong le, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+osteoclast,+bone+remodelling+and+treatment+of+metabolic+bone+disease.">The osteoclast, bone remodelling and treatment of metabolic bone disease.</a> European journal of clinical investigation. 42, 1332-1341 (2012).</li><li>Lacey, D. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoprotegerin+ligand+is+a+cytokine+that+regulates+osteoclast+differentiation+and+activation.">Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation.</a> Cell. 93, 165-176 (1998).</li><li>Mizuno, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+mice+overexpressing+soluble+osteoclast+differentiation+factor+(sODF)+exhibit+severe+osteoporosis.">Transgenic mice overexpressing soluble osteoclast differentiation factor (sODF) exhibit severe osteoporosis.</a> Journal of bone and mineral metabolism. 20, 337-344 (2002).</li><li>Bucay, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=osteoprotegerin-deficient+mice+develop+early+onset+osteoporosis+and+arterial+calcification.">osteoprotegerin-deficient mice develop early onset osteoporosis and arterial calcification.</a> Gene., &amp; development. 12, 1260-1268 (1998).</li><li>Suda, T., Liu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamic+gene+delivery:+its+principles+and+applications.">Hydrodynamic gene delivery: its principles and applications.</a> Molecular therapy: the journal of the American Society of Gene Therapy. 15, 2063-2069 (2007).</li><li>Wronski, T. J., Dann, L. M., Scott, K. S., Cintron, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+effects+of+ovariectomy+and+aging+on+the+rat+skeleton.">Long-term effects of ovariectomy and aging on the rat skeleton.</a> Calcified tissue international. 45, 360-366 (1989).</li><li>Seto, H., Aoki, K., Kasugai, S., Ohya, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trabecular+bone+turnover,+bone+marrow+cell+development,+and+gene+expression+of+bone+matrix+proteins+after+low+calcium+feeding+in+rats.">Trabecular bone turnover, bone marrow cell development, and gene expression of bone matrix proteins after low calcium feeding in rats.</a> Bone. 25, 687-695 (1999).</li><li>Lelovas, P. P., Xanthos, T. T., Thoma, S. E., Lyritis, G. P., Dontas, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+laboratory+rat+as+an+animal+model+for+osteoporosis+research.">The laboratory rat as an animal model for osteoporosis research.</a> Comparative medicine. 58, 424-430 (2008).</li><li>Sherman, B. M., West, J. H., Korenman, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+menopausal+transition:+analysis+of+LH,+FSH,+estradiol,+and+progesterone+concentrations+during+menstrual+cycles+of+older+women.">The menopausal transition: analysis of LH, FSH, estradiol, and progesterone concentrations during menstrual cycles of older women.</a> The Journal of clinical endocrinology and metabolism. 42, 629-636 (1976).</li><li>Hughes, D. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+promotes+apoptosis+of+murine+osteoclasts+mediated+by+TGF-beta.">Estrogen promotes apoptosis of murine osteoclasts mediated by TGF-beta.</a> Nature medicine. 2, 1132-1136 (1996).</li><li>Kousteni, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nongenotropic,+sex-nonspecific+signaling+through+the+estrogen+or+androgen+receptors:+dissociation+from+transcriptional+activity.">Nongenotropic, sex-nonspecific signaling through the estrogen or androgen receptors: dissociation from transcriptional activity.</a> Cell. 104, 719-730 (2001).</li><li>Ominsky, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RANKL+inhibition+with+osteoprotegerin+increases+bone+strength+by+improving+cortical+and+trabecular+bone+architecture+in+ovariectomized+rats.">RANKL inhibition with osteoprotegerin increases bone strength by improving cortical and trabecular bone architecture in ovariectomized rats.</a> Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 23, 672-682 (2008).</li><li>Kitazawa, R., Kimble, R. B., Vannice, J. L., Kung, V. T., Pacifici, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-1+receptor+antagonist+and+tumor+necrosis+factor+binding+protein+decrease+osteoclast+formation+and+bone+resorption+in+ovariectomized+mice.">Interleukin-1 receptor antagonist and tumor necrosis factor binding protein decrease osteoclast formation and bone resorption in ovariectomized mice.</a> The Journal of clinical investigation. 94, 2397-2406 (1994).</li><li>Weitzmann, M. N., Pacifici, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+deficiency+and+bone+loss:+an+inflammatory+tale.">Estrogen deficiency and bone loss: an inflammatory tale.</a> The Journal of clinical investigation. 116, 1186-1194 (2006).</li><li>Suda, T., Nakamura, I., Jimi, E., Takahashi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+osteoclast+function.">Regulation of osteoclast function.</a> J Bone Miner Res. 12, 869-879 (1997).</li><li>Asagiri, M., Takayanagi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+understanding+of+osteoclast+differentiation.">The molecular understanding of osteoclast differentiation.</a> Bone. 40, 251-264 (2007).</li><li>Matsuzaki, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoclast+differentiation+factor+(ODF)+induces+osteoclast-like+cell+formation+in+human+peripheral+blood+mononuclear+cell+cultures.">Osteoclast differentiation factor (ODF) induces osteoclast-like cell formation in human peripheral blood mononuclear cell cultures.</a> Biochemical and biophysical research communications. 246, 199-204 (1998).</li><li>Adamopoulos, I. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synovial+fluid+macrophages+are+capable+of+osteoclast+formation+and+resorption.">Synovial fluid macrophages are capable of osteoclast formation and resorption.</a> The Journal of pathology. 208, 35-43 (2006).</li><li>Adamopoulos, I. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-17A+upregulates+receptor+activator+of+NF-kappaB+on+osteoclast+precursors.">Interleukin-17A upregulates receptor activator of NF-kappaB on osteoclast precursors.</a> Arthritis researc., &amp; therapy. 12, (2010).</li><li>Jones, D., Glimcher, L. H., Aliprantis, A. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoimmunology+at+the+nexus+of+arthritis,+osteoporosis,+cancer,+and+infection.">Osteoimmunology at the nexus of arthritis, osteoporosis, cancer, and infection.</a> J Clin Invest. 121, 2534-2542 (2011).</li><li>Sato, K., Takayanagi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoclasts,+rheumatoid+arthritis,+and+osteoimmunology.">Osteoclasts, rheumatoid arthritis, and osteoimmunology.</a> Curr Opin Rheumatol. 18, 419-426 (2006).</li><li>Das, S., Crockett, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoporosis+-+a+current+view+of+pharmacological+prevention+and+treatment.">Osteoporosis - a current view of pharmacological prevention and treatment.</a> Drug design, development and therapy. 7, 435-448 (2013).</li><li>Chen, Z. Y., He, C. Y., Ehrhardt, A., Kay, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minicircle+DNA+vectors+devoid+of+bacterial+DNA+result+in+persistent+and+high-level+transgene+expression+in+vivo.">Minicircle DNA vectors devoid of bacterial DNA result in persistent and high-level transgene expression in vivo.</a> Molecular therapy : the journal of the American Society of Gene Therapy. 8, 495-500 (2003).</li><li>Kay, M. A., He, C. Y., Chen, Z. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+robust+system+for+production+of+minicircle+DNA+vectors.">A robust system for production of minicircle DNA vectors.</a> Nature biotechnology. 28, 1287-1289 (2010).</li><li>Chen, Z. Y., He, C. Y., Kay, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+production+and+purification+of+minicircle+DNA+vector+free+of+plasmid+bacterial+sequences+and+capable+of+persistent+transgene+expression+in+vivo.">Improved production and purification of minicircle DNA vector free of plasmid bacterial sequences and capable of persistent transgene expression in vivo.</a> Human gene therapy. 16, 126-131 (2005).</li><li>Halleen, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tartrate-resistant+acid+phosphatase+5b:+a+novel+serum+marker+of+bone+resorption.">Tartrate-resistant acid phosphatase 5b: a novel serum marker of bone resorption.</a> Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 15, 1337-1345 (2000).</li><li>Adamopoulos, I. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-23+is+critical+for+induction+of+arthritis,+osteoclast+formation,+and+maintenance+of+bone+mass.">IL-23 is critical for induction of arthritis, osteoclast formation, and maintenance of bone mass.</a> J Immunol. 187, 951-959 (2011).</li><li>Suda, T., Takahashi, N., Martin, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+osteoclast+differentiation.">Modulation of osteoclast differentiation.</a> Endocrine reviews. 13, 66-80 (1992).</li><li>Takahashi, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoblastic+cells+are+involved+in+osteoclast+formation.">Osteoblastic cells are involved in osteoclast formation.</a> Endocrinology. 123, 2600-2602 (1988).</li><li>Bradley, E. W., Oursler, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoclast+culture+and+resorption+assays.">Osteoclast culture and resorption assays.</a> Methods Mol Biol. 455, 19-35 (2008).</li><li>Arai, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Commitment+and+differentiation+of+osteoclast+precursor+cells+by+the+sequential+expression+of+c-Fms+and+receptor+activator+of+nuclear+factor+kappaB+(RANK)+receptors.">Commitment and differentiation of osteoclast precursor cells by the sequential expression of c-Fms and receptor activator of nuclear factor kappaB (RANK) receptors.</a> The Journal of experimental medicine. 190, 1741-1754 (1999).</li><li>Fuller, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+colony-stimulating+factor+stimulates+survival+and+chemotactic+behavior+in+isolated+osteoclasts.">Macrophage colony-stimulating factor stimulates survival and chemotactic behavior in isolated osteoclasts.</a> The Journal of experimental medicin. 178, 1733-1744 (1993).</li><li>Edwards, J. R., Mundy, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+osteoclast+biology:+old+findings+and+new+insights+from+mouse+models.">Advances in osteoclast biology: old findings and new insights from mouse models.</a> Nature reviews. Rheumatology. 7, 235-243 (2011).</li><li>Weinstein, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promotion+of+osteoclast+survival+and+antagonism+of+bisphosphonate-induced+osteoclast+apoptosis+by+glucocorticoids.">Promotion of osteoclast survival and antagonism of bisphosphonate-induced osteoclast apoptosis by glucocorticoids.</a> The Journal of clinical investigation. 109, 1041-1048 (2002).</li></ol>

The authors have nothing to disclose.

肌肉骨骼条件，导致发病率和致残的主要原因，并包含超过150个疾病和证候;影响大约90万美国人。关节的炎症和骨质破坏是骨骼肌肉条件的主要功能，包括关节炎和骨质疏松症。骨质疏松症是骨弱化完整性，常常导致骨的骨折的情况。关节炎是一种慢性的，衰弱的疾病，其特征是变得肿胀，招标和僵硬，限制正常运动，并可能导致残疾的关节发炎。虽然肌肉骨骼疾病的发病机制大大不同于彼此，?...

肌肉骨骼疾病影响到千百万人在美国和现在的严重后果为国家和地方卫生系统1。这些疾病的特点是骨损失和关节功能，需要广泛的治疗和恢复很长时间。通常，在破骨细胞的数量和/或活性的相对增加，细胞专门的重吸收骨骼，在骨质疏松和关节炎，观察2。在生理条件下的破骨细胞的数量和活性是通过核因子κ-B配体（RANKL），它是由成骨细胞产生的受体活化调节。护骨素（OPG），诱饵受体RANKL由成骨细胞3也产生涉及sRANKL全身过度表达，或删除OPG的体内动物模型是非常有价值的骨质疏松症的研究。然而，这些方法需要转基因小鼠4,5的产生。这里，一种新型的替代过表达sRANKL为肌肉骨骼相关的疾病的研究的方法进行说明。具体地，小环（MC）DNA技术和流体动力传递方法被用来实现sRANKL的体内基因转移和过表达小鼠sRANKL全身6。 这种方法也补充其他体内模型的骨质疏松，如由低钙饮食8以下卵巢切除7和饮食干预破骨细胞的激素调节。这些模型是研究的但是他们需要外科手术，可能需要长达数月，在一个显著的成本9肌肉骨骼相关疾病的不同方面非常有用。卵巢切除（OVX）啮齿动物模型的实验动物模型，其中卵巢切除导致雌激素缺乏，从而模仿人类绝经后骨质疏松症10。人绝经期后骨质疏松症，一个条件，其中的雌激素deficiENCY导致骨折的风险增加和骨质疏松症影响约800万妇女在美国就有。虽然绝经后骨质疏松症的去势模型是非常有用的它提供了有限的优势，在一般的研究骨质疏松症。雌激素可以抑制骨质流失，通过诱导破骨细胞，抑制成骨细胞凋亡，因此，在其没有增加破骨细胞活性观察10-12。一个RANKL-OPG比例失衡有利于骨吸收也观察到13。然而， 在体内的雌激素缺乏也伴随着降低的转化生长因子β（TGFβ），增加白细胞介素-7（IL-7）和肿瘤坏死因子，IL-1和IL-6的水平14,15。由于这些细胞因子已知的骨重塑调节功能独立于RANKL通路，这是不可能的属性的任何破骨细胞的活化，仅与RANKL-RANK轴。在本文中描述的模型使研究人员在VIV研究ØRANKL-RANK轴的破骨细胞和骨量丢失无促炎细胞因子相比，去卵巢啮齿类动物模型。 此外， 体外破骨细胞技术是必不可少的工具来研究破骨细胞活化肌肉骨骼疾病的潜在治疗方法。以前的研究还表明，与小鼠巨噬细胞集落刺激因子（M-CSF）和小鼠sRANKL培养小鼠骨髓衍生的巨噬细胞（桥梁养护）能导致破骨细胞分化3,16,17。在此，协议来生成从小鼠骨髓以及从人外周血单核细胞（PBMC） 在体外 18多核破骨细胞样细胞进行了描述。都定义一个成熟的终末分化和功能齐全的破骨细胞所需的基于细胞的测定也简单说明。这些体外技术补充体内的方法小说一起用于为powerful调查工具，研究破骨细胞分化和活化。使用这些系统中，科学家们能够产生在体内和体外的破骨细胞和确定所需的增殖和活化的刺激和信号，以及测试的药理和生物抑制剂的功效。

<table border="0" cellpadding="0" cellspacing="0" width="677">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:320px;">alpha-MEM</td>
			<td style="width:71px;">Life Technologies&#160;</td>
			<td style="width:119px;">12561-056</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">Human M-CSF</td>
			<td style="width:71px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-096-492</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">Mouse M-CSF</td>
			<td style="width:71px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-094-643</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">Human RANK-Ligand - soluble</td>
			<td style="width:71px;">Miltenyi Biotec</td>
			<td>130-094-631</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">Mouse RANK-Ligand - soluble</td>
			<td style="width:71px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-094-076</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Tailveiner Restrainer for mice</td>
			<td style="width:71px;">Braintree</td>
			<td style="width:119px;">TV-150 STD</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Mouse TRANCE/RANK L/TNFSF11 Quantikine ELISA Kit&#160;</td>
			<td style="width:71px;">R&#38;D systems</td>
			<td style="width:119px;">MTR00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acid Phosphatase, Leukocyte (TRAP) Kit</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">387A</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:320px;">MouseTRAP assay&#160;</td>
			<td style="width:71px;">immunodiagnostic systems</td>
			<td style="width:119px;">SB-TR103</td>
			<td></td>
		</tr>
	</tbody>
</table>

a novel in vivo gene transfer technique and in vitro cell based assays for the study of bone loss in musculoskeletal disorders

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone resorption. Osteoclasts can be generated in vitro from monocyte/macrophage precursor cells in the presence of certain cytokines, which promote survival and differentiation. Here, both in vivo and in vitro techniques are demonstrated, which allow scientists to study different cytokine contributions towards osteoclast differentiation, signaling, and activation. The minicircle DNA delivery gene transfer system provides an alternative method to establish an osteoporosis-related model is particularly useful to study the efficacy of various pharmacological inhibitors in vivo. Similarly, in vitro culturing protocols for producing osteoclasts from human precursor cells in the presence of specific cytokines enables scientists to study osteoclastogenesis in human cells for translational applications. Combined, these techniques have the potential to accelerate drug discovery efforts for osteoclast-specific targeted therapeutics, which may benefit millions of osteoporosis and arthritis patients worldwide.

这里， 在体内和细胞培养协议破骨细胞分化的新基因转移技术，用于差分前体细胞在体外破骨细胞作为研究细胞因子对破骨细胞形成的影响的方法进行了描述。在图1中，绿色荧光蛋白和小鼠sRANKL MC小鼠成功的基因转移的代表性结果示。在图2中，示出的小鼠骨髓或前体细胞向破骨细胞通过明场显微术的人PBMC细胞分化时程文化的代表性图像， 图3?...

Watch this Scientific Journal Video about A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders at JoVE.com

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described.

一种新型的<em&gt;在体内</em&gt;基因转移技术和<em&gt;在体外</em&gt;细胞分析为肌肉骨骼疾病骨质流失的研究

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone ...

a-novel-vivo-gene-transfer-technique-vitro-cell-based-assays-for

Research

JoVE Journal

Medicine

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

11.6K 次观看.  University of California, Davis. Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described. 