この研究は、中国国立自然科学財団（81573992）によって支持された。私たちはEmily K. LoとKathleen DiNapoliの言語編集支援に感謝します。

このプロトコルは上海漢方医学の動物飼育指針に従っており、動物実験上海動物倫理委員会の承認を受けています。 1.プロトコルI：EXDの調製<ol><li> 計算 <ol><li> 6ヵ月齢の女性刷本管理領域（ICR）マウス（0.02kg /マウス）10匹の治療群にEXDを投与し、1週間あたり5日間12週間投与する。 注：合計1.702 EXDが必要です。 EXDは離散的な増分で測定されるため、実際には2つのEXDを管理します。 </li><li>マウス1匹につき適用されるEXDの量を計算する（ すなわち、 V =（0.1418 EXD / kg・日）*（0.02kg /マウス）* 50mL / EXD = 0.14mL /マウス・日）。 </li></ol></li><li> 浸漬 <ol><li> 2つのEXDのすべての原材料（ すなわち、Curculigo orchioides Gaertn （18g）、 Herba Epimedii （18g）、 Radix Morindae Officinalis （18g）、 R（18g）、 Cortex Phellodendri （12g）、 Rhizoma Anemarrhenae （12g）、合計96g）を蓋付き容器に入れた。 注：セラミック容器を推奨します。 </li><li>生ハーブを500mLの蒸留水で1時間洗浄します。水はハーブを約1インチ覆うべきです。すべてのハーブを徹底的に浸しましょう。 </li></ol></li><li> 最初の水の煎じ薬 <ol><li>水が沸騰するまで（約5〜10分;加熱時間、容器、およびハーブと水の量に依存します）、ガス炊飯器または誘導加熱調理器を使用してハーブを高熱で加熱します。低煮沸の電源を2時間倒します。 </li></ol></li><li> 最初のろ過 <ol><li>定期的なろ紙またはガーゼと綿で500 mLガラスビーカーを覆う。フィルターを通して、煎じ薬を慎重にビーカーに注ぎます。容器にハーブを残す。 </li><li>残った薬草残渣を返す容器にろ紙を移す。 </li></ol></li><li> 2回目の水煎じ <ol><li>ハーブで蒸留水を容器に加えます。ハーブを約1インチ覆うようにしましょう。 </li><li>手順1.3を繰り返します。 </li></ol></li><li> 二次ろ過 <ol><li>ステップ1.4.1と1.4.2を繰り返します。 </li><li> 2回目の煎じ薬を同じビーカーに注ぎます。 1回目と2回目の煎じ薬を混ぜる。 </li></ol></li><li> 濃度 <ol><li>ビーカーをアスベストフリーのワイヤーガーゼを挟んでガス炊飯器の上に置き、低煮沸器で加熱し、ガラス棒でゆっくりと連続的に攪拌する。 </li><li>煎剤が200 mLになったら、500 mLのビーカーに移す。 </li><li>煎じ薬が100mLになるまでステップ1.7.1を繰り返します。 </li></ol></li><li> 保管と管理 <ol><li>濃縮した煎じ薬を滅菌ガラス瓶に移す。それは室温に冷やしてみましょう再（RT）。 1週間以内に使用する場合は4℃で、長期間保存する場合は-70℃で保管してください。 </li><li>強制栄養針を用いて、1日1回、1週間に5日、12週間、卵巣摘出（OVX）マウスに煎じ薬0.14mL /マウスを投与する。 </li></ol></li></ol> 2.プロトコルII：EXD含有血清の調製<ol><li> 計算 <ol><li>各ラット（1ヶ月齢）の体重として0.15kgを用いて、ラット相当用量（RED）を計算する：dB =（1/60）*（90/100）*（60 / 0.15） 1/3 = 0.111 EXD / kg /日。 注：100mLの培地（10％EXD含有血清）を調製するためには、合計10mLのEXD含有血清が必要です。各ラットは2mLの血清を提供すると予想される。したがって、EXD投与には1日1回3日間、6匹のラット（20％の損失を考慮する）が必要である。 EXDの数は、（0.111 EXD / kg・日）*（0.15kg /ラット）*（6匹のラット）*（3日）= 0.300 EXD。再び、EXDは離散的な増分で測定されるので、1 EXDを使用します。 </li><li>ラット（ すなわち、 V =（0.111 EXD / kg・日）*（0.15kg /ラット）*（50mL / EXD）= 0.83mL /ラット・日）に適用されるEXDの容量を計算する。 </li></ol></li><li> 浸漬 <ol><li> 1 EXD用の原料を蓋付き容器に入れます。生ハーブを蒸留水に1時間浸します。水はハーブを約1インチ覆うべきです。すべてのハーブを徹底的に浸しましょう。 </li></ol></li><li> 最初の水の煎じ薬 <ol><li>水が沸騰するまで（約5〜10分;時間は熱出力、容器、およびハーブと水の量に依存します）、誘導加熱器を高出力で使用してハーブを加熱します。電源を2時間低煮沸にします。 </li></ol></li><li> 最初のろ過 <ol><li>定期的なろ紙またはガーゼと綿で500 mLガラスビーカーを覆う。フィルターを通して、煎じ薬を慎重にビーカーに注ぎます。容器にハーブを残す。 </li><li&gt;ろ紙上のハーブ残渣を容器に戻します。 </li></ol></li><li> 2回目の水煎じ <ol><li>ハーブで蒸留水を容器に加えます。ハーブを約1インチ覆うようにしましょう。 </li><li>ステップ2.4を繰り返します。 </li></ol></li><li> 二次ろ過 <ol><li>手順2.5.1と2.5.2を繰り返します。 </li><li>ステップ2.4.1と同じビーカーに2回目の煎じ薬を注ぎ、1回目と2回目の煎じ薬を混ぜます。 </li></ol></li><li> 濃度 <ol><li>ビーカーをアスベストフリーのワイヤーガーゼを挟んだガス炊飯器に入れ、低煮沸器で加熱し、ガラス棒でゆっくりと連続的に攪拌する。 </li><li>煎じ薬が100mLになったら、100mLビーカーに移す。 </li><li>煎じ薬が50mLになるまで2.7.1の手順を繰り返します。 </li></ol></li><li> 保管と管理 <ol><li>濃縮煎じ薬をステーに移すガラス瓶。それをRTに冷やす。 1週間以内に使用する場合は4℃で、長期間保存する場合は-70℃で保管してください。 </li><li> 1日1回、0.83mL /ラットを3日間胃内投与する。 </li></ol></li><li> EXD含有血清調製物 <ol><li>最後のEXD投与の1時間後に300mg / 100gの80mg / kgケタミンおよび10mg / kgキシラジンを腹腔内注射することによりラットを麻酔する。つま先で適切な麻酔を確認してください。 </li><li>メスを使用して腹部から胸郭の底にラットの皮膚と腹膜を切開する（またはまっすぐに操作するはさみ）。それぞれ約5cmと0.5cmの長さと深さの切開を作成します。ティッシュペーパーを使用して腹部内臓を左に動かします。 </li><li>ティシュペーパーを使用して、腹部大動脈の結合組織を除去して、血管をはっきりと露出させる。 </li><li> 10mL、22ゲージのシリンジを使用して、腹部大動脈からゆっくりと血液を採取する。それを転送する針を除去した後の15mLの滅菌チューブ; 1匹のラットは8〜10mLの血液を産生することができる。 注：採血が始まるとラットは生きている（ すなわち、腹部大動脈が拍動する）。 </li><li>室温RTで30〜60分間、直立姿勢で血液を凝固させる。 500-600×gで20分間遠心分離する。すべての上清（血清）を注意深く1つの50mLの滅菌チューブに入れ、すべての血清（異なる動物由来）を一緒に混合する。 </li><li>血清を56℃の水浴中で30分間インキュベートすることにより熱不活性化を行う。 0.22μmの孔径の親水性ポリエーテルスルホン膜を有するシリンジフィルターを用いて血清を濾過する。新鮮な血清を使用するか、-20℃で保存してください。 </li></ol></li><li> 対照薬物含有血清調製物（対照群において使用される） <ol><li> 1日1回3日間同じ容量の生理食塩水（0.83mL）で各ラットを投与する。他のステップはステップ2.9と同じです。</li></ol></li><li> 応用 <ol><li> EXD含有血清または薬物含有血清10mLを各100mLの培地に加え、1％ペニシリン - ストレプトマイシン（PS） 23に加える。 </li></ol></li></ol>

<ol>
	<li>Zhang, L. G., Ouyang, X. W., Wu, T. T., Ni, L. J., Shi, W. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+evaluation+of+in+vitro+effects+and+interactions+of+active+fractions+in+a+Chinese+medicinal+formula+(Yaotongning+Capsule)+on+rat+chondrocytes.">Quantitative evaluation of in vitro effects and interactions of active fractions in a Chinese medicinal formula (Yaotongning Capsule) on rat chondrocytes.</a> J Ethnopharmacol. 155 (3), 1424-1432 (2014).</li><li>Ogawa, Y., Fujii, Y., Sugiyama, R., Konishi, 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+role+of+the+seven+crude+drug+components+in+the+sleep-promoting+effect+of+Yokukansan.">The role of the seven crude drug components in the sleep-promoting effect of Yokukansan.</a> J Ethnopharmacol. 177, 19-27 (2016).</li><li>Kim, J. H., Doh, E. J., Lee, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+Medicinal+Categorization+of+Atractylodes+japonica+Koidz.+by+Using+Internal+Transcribed+Spacer+Sequencing+Analysis+and+HPLC+Fingerprinting+Combined+with+Statistical+Tools.">Evaluation of Medicinal Categorization of Atractylodes japonica Koidz. by Using Internal Transcribed Spacer Sequencing Analysis and HPLC Fingerprinting Combined with Statistical Tools.</a> Evid Based Complement Alternat Med. 2016, 2926819 (2016).</li><li>Sheridan, H., 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=The+potential+of+metabolic+fingerprinting+as+a+tool+for+the+modernisation+of+TCM+preparations.">The potential of metabolic fingerprinting as a tool for the modernisation of TCM preparations.</a> J Ethnopharmacol. 140 (3), 482-491 (2012).</li><li>Planas, G. M., Kucacute, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contraceptive+Properties+of+Stevia+rebaudiana.">Contraceptive Properties of Stevia rebaudiana.</a> Science. 162 (3857), 1007 (1968).</li><li>Tashino, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Serum+pharmacology"+and+"serum+pharmaceutical+chemistry":+from+pharmacology+of+Chinese+traditional+medicines+to+start+a+new+measurement+of+drug+concentration+in+blood.">"Serum pharmacology" and "serum pharmaceutical chemistry": from pharmacology of Chinese traditional medicines to start a new measurement of drug concentration in blood.</a> Ther Drug Monit Res. 5, 54-64 (1988).</li><li>Cao, Y., Liu, F., Huang, Z., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+effects+of+Guanxin+Shutong+capsule+drug-containing+serum+on+tumor+necrosis+factor-alpha-induced+endothelial+dysfunction+through+nicotinamide+adenine+dinucleotide+phosphate+oxidase+and+the+nitric+oxide+pathway.">Protective effects of Guanxin Shutong capsule drug-containing serum on tumor necrosis factor-alpha-induced endothelial dysfunction through nicotinamide adenine dinucleotide phosphate oxidase and the nitric oxide pathway.</a> Exp Ther Med. 8 (3), 998-1004 (2014).</li><li>Fu, 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=Ex+Vivo+Stromal+Cell-Derived+Factor+1-Mediated+Differentiation+of+Mouse+Bone+Marrow+Mesenchymal+Stem+Cells+into+Hepatocytes+Is+Enhanced+by+Chinese+Medicine+Yiguanjian+Drug-Containing+Serum.">Ex Vivo Stromal Cell-Derived Factor 1-Mediated Differentiation of Mouse Bone Marrow Mesenchymal Stem Cells into Hepatocytes Is Enhanced by Chinese Medicine Yiguanjian Drug-Containing Serum.</a> Evid Based Complement Alternat Med. 2016, 7380439 (2016).</li><li>Chen, X., 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=Application+of+serum+pharmacology+in+evaluating+the+antitumor+effect+of+Fuzheng+Yiliu+Decoction+from+Chinese+Medicine.">Application of serum pharmacology in evaluating the antitumor effect of Fuzheng Yiliu Decoction from Chinese Medicine.</a> Chin J Integr Med. 20 (6), 450-455 (2014).</li><li>Sze, S. C., 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=A+novel+mechanism:+Erxian+Decoction,+a+Chinese+medicine+formula,+for+relieving+menopausal+syndrome.">A novel mechanism: Erxian Decoction, a Chinese medicine formula, for relieving menopausal syndrome.</a> J Ethnopharmacol. 123 (1), 27-33 (2009).</li><li>Sze, S. C., 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=Effects+of+Erxian+decoction,+a+Chinese+medicinal+formulation,+on+serum+lipid+profile+in+a+rat+model+of+menopause.">Effects of Erxian decoction, a Chinese medicinal formulation, on serum lipid profile in a rat model of menopause.</a> Chin Med. 6, 40 (2011).</li><li>Zhong, L. 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=A+randomized,+double-blind,+controlled+trial+of+a+Chinese+herbal+formula+(Er-Xian+decoction)+for+menopausal+symptoms+in+Hong+Kong+perimenopausal+women.">A randomized, double-blind, controlled trial of a Chinese herbal formula (Er-Xian decoction) for menopausal symptoms in Hong Kong perimenopausal women.</a> Menopause. 20 (7), 767-776 (2013).</li><li>Wang, S. W., 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=Steroidogenic+effect+of+Erxian+decoction+for+relieving+menopause+via+the+p-Akt/PKB+pathway+in+vitro+and+in+vivo.">Steroidogenic effect of Erxian decoction for relieving menopause via the p-Akt/PKB pathway in vitro and in vivo.</a> J Ethnopharmacol. , (2016).</li><li>Lin, L., Wu, S., Tang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[Clinical+observation+and+experimental+study+of+the+treatment+of+aplastic+anemia+by+warming+and+tonifying+the+spleen+and+kidney].">[Clinical observation and experimental study of the treatment of aplastic anemia by warming and tonifying the spleen and kidney].</a> Zhong Xi Yi Jie He Za Zhi. 10 (5), 272-274 (1990).</li><li>Nian, H., 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=Antiosteoporotic+activity+of+Er-Xian+Decoction,+a+traditional+Chinese+herbal+formula,+in+ovariectomized+rats.">Antiosteoporotic activity of Er-Xian Decoction, a traditional Chinese herbal formula, in ovariectomized rats.</a> J Ethnopharmacol. 108 (1), 96-102 (2006).</li><li>Qin, 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=Antiosteoporotic+chemical+constituents+from+Er-Xian+Decoction,+a+traditional+Chinese+herbal+formula.">Antiosteoporotic chemical constituents from Er-Xian Decoction, a traditional Chinese herbal formula.</a> J Ethnopharmacol. 118 (2), 271-279 (2008).</li><li>Xue, 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=A+HNMR-based+metabonomics+study+of+postmenopausal+osteoporosis+and+intervention+effects+of+Er-Xian+Decoction+in+ovariectomized+rats.">A HNMR-based metabonomics study of postmenopausal osteoporosis and intervention effects of Er-Xian Decoction in ovariectomized rats.</a> Int J Mol Sci. 12 (11), 7635-7651 (2011).</li><li>Lu, X. N., Xu, X. R., Lin, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[Clinical+observation+of+bushen+er'xian+decoction+in+treating+premature+ovarian+failure].">[Clinical observation of bushen er'xian decoction in treating premature ovarian failure].</a> Zhongguo Zhong Xi Yi Jie He Za Zhi. 28 (7), 594-596 (2008).</li><li>Yu, X., 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=Anti-angiogenic+activity+of+Erxian+Decoction,+a+traditional+Chinese+herbal+formula,+in+zebrafish.">Anti-angiogenic activity of Erxian Decoction, a traditional Chinese herbal formula, in zebrafish.</a> Biol Pharm Bull. 35 (12), 2119-2127 (2012).</li><li>Chu, E. 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=An+in+vitro+and+in+vivo+investigation+of+the+antimetastatic+effects+of+a+Chinese+medicinal+decoction,+erxian+decoction,+on+human+ovarian+cancer+models.">An in vitro and in vivo investigation of the antimetastatic effects of a Chinese medicinal decoction, erxian decoction, on human ovarian cancer models.</a> Integr Cancer Ther. 12 (4), 336-346 (2013).</li><li>Zhu, Z., Li, L., Jin, X., Fang, J., Zhang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Er-Xian+Decoction,+a+traditional+Chinese+herbal+formula,+intervening+early+in+hypothalamic-pituitary+axis+of+male+rats+with+delayed+puberty.">Er-Xian Decoction, a traditional Chinese herbal formula, intervening early in hypothalamic-pituitary axis of male rats with delayed puberty.</a> Pharmacogn Mag. 10 (40), 517-521 (2014).</li><li>Miao, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Experimental animals and technology. 1997, 145 (1997).</li><li>Soleimani, M., Nadri, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+isolation+and+culture+of+mesenchymal+stem+cells+from+mouse+bone+marrow.">A protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow.</a> Nat Protoc. 4 (1), 102-106 (2009).</li><li>Lu, S., Huang, J., Wang, J., 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=Er-Xian+Decoction+Stimulates+Osteoblastic+Differentiation+of+Bone+Mesenchymal+Stem+Cells+in+Ovariectomized+Mice+and+Its+Gene+Profile+Analysis.">Er-Xian Decoction Stimulates Osteoblastic Differentiation of Bone Mesenchymal Stem Cells in Ovariectomized Mice and Its Gene Profile Analysis.</a> Stem Cells International. 2016, 4079210 (2016).</li><li>Turner, P. V., Brabb, T., Pekow, C., Vasbinder, 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=Administration+of+substances+to+laboratory+animals:+routes+of+administration+and+factors+to+consider.">Administration of substances to laboratory animals: routes of administration and factors to consider.</a> J Am Assoc Lab Anim Sci. 50 (5), 600-613 (2011).</li><li>Wu, C., 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=Simultaneous+determination+of+seven+flavonoids+in+dog+plasma+by+ultra-performance+liquid+chromatography-tandem+mass+spectrometry+and+its+application+to+a+bioequivalence+study+of+bioactive+components+in+Herba+Epimedii+and+Er-Xian+Decoction.">Simultaneous determination of seven flavonoids in dog plasma by ultra-performance liquid chromatography-tandem mass spectrometry and its application to a bioequivalence study of bioactive components in Herba Epimedii and Er-Xian Decoction.</a> J Pharm Biomed Anal. 54 (1), 186-191 (2011).</li><li>Hu, Y. 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=Identification+of+the+major+chemical+constituents+and+their+metabolites+in+rat+plasma+and+various+organs+after+oral+administration+of+effective+Erxian+Decoction+(EXD)+fraction+by+liquid+chromatography-mass+spectrometry.">Identification of the major chemical constituents and their metabolites in rat plasma and various organs after oral administration of effective Erxian Decoction (EXD) fraction by liquid chromatography-mass spectrometry.</a> Biomed Chromatogr. 24 (5), 479-489 (2010).</li><li>Xue, 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=Effects+and+interaction+of+icariin,+curculigoside,+and+berberine+in+er-xian+decoction,+a+traditional+chinese+medicinal+formula,+on+osteoclastic+bone+resorption.">Effects and interaction of icariin, curculigoside, and berberine in er-xian decoction, a traditional chinese medicinal formula, on osteoclastic bone resorption.</a> Evid Based Complement Alternat Med. , (2012).</li><li>Nair, A. B., Jacob, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+practice+guide+for+dose+conversion+between+animals+and+human.">A simple practice guide for dose conversion between animals and human.</a> J Basic Clin Pharm. 7 (2), 27-31 (2016).</li><li>Xu, X., 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=Protective+effect+of+the+traditional+Chinese+medicine+xuesaitong+on+intestinal+ischemia-reperfusion+injury+in+rats.">Protective effect of the traditional Chinese medicine xuesaitong on intestinal ischemia-reperfusion injury in rats.</a> Int J Clin Exp Med. 8 (2), 1768-1779 (2015).</li><li>Li, Z., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+methods+for+Chinese+herbs+serum+pharmcology.">On the methods for Chinese herbs serum pharmcology.</a> Zhong Guo Zhong Yi Yao Xing Xi Za Zhi. 9 (2), 5-6 (2002).</li><li>Jiang, Y. R., 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=Effect+of+chinese+herbal+drug-containing+serum+for+activating-blood+and+dispelling-toxin+on+ox-LDL-induced+inflammatory+factors'+expression+in+endothelial+cells.">Effect of chinese herbal drug-containing serum for activating-blood and dispelling-toxin on ox-LDL-induced inflammatory factors' expression in endothelial cells.</a> Chin J Integr Med. 18 (1), 30-33 (2012).</li><li>Guo, C. Y., Ma, X. J., Liu, Q., Yin, H. J., Shi, D. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[Effect+of+Chinese+herbal+drug-containing+serum+for+activating+blood,+activating+blood+and+dispelling+toxin+on+TNF-alpha-induced+adherence+between+endothelial+cells+and+neutrophils+and+the+expression+of+MAPK+pathway].">[Effect of Chinese herbal drug-containing serum for activating blood, activating blood and dispelling toxin on TNF-alpha-induced adherence between endothelial cells and neutrophils and the expression of MAPK pathway].</a> Zhongguo Zhong Xi Yi Jie He Za Zhi. 35 (2), 204-209 (2015).</li><li>Li, Y., Xia, J. Y., Chen, W., Deng, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Ling+Qi+Juan+Gan+capsule+drug-containing+serum+on+PDGF-induced+proliferation+and+JAK/STAT+signaling+of+HSC-T6+cells].">Effects of Ling Qi Juan Gan capsule drug-containing serum on PDGF-induced proliferation and JAK/STAT signaling of HSC-T6 cells].</a> Zhonghua Gan Zang Bing Za Zhi. 21 (9), 663-667 (2013).</li><li>Li, Y. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[Some+issues+in+methology+of+Chinese+herbs+serum+pharmcology].">[Some issues in methology of Chinese herbs serum pharmcology].</a> Zhong Yao Xin Yao Yu Lin Chuang Yao Li. 10 (5), 263 (1999).</li><li>Zhang, 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=[A+review+of+Chinese+herbs+serum+pharmcology+methodological+study].">[A review of Chinese herbs serum pharmcology methodological study].</a> Nan Jing Zhong Yi Yao Da Xue Xue Bao. 18 (4), 254 (2002).</li><li>Zhang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[Issues+and+strategies+for+study+of+serum+pharmcology+in+oncology].">[Issues and strategies for study of serum pharmcology in oncology].</a> Zhong Yi Yan Jiu. 17 (5), 13-14 (2004).</li></ol>

著者らは、競合する金銭的利益がないと宣言している。

近年、東アジアの臨床現場で何千年も使用されてきた代替薬の一種であるハーブ薬が注目されています。伝統的な漢方薬は、現代医学の「ベンチ・ベッド」のパターンとは異なり、まずそのメカニズムを説明するために「ベッド・ツー・ベンチ」のパターンが必要です。これに続いて、ベンチ段階で行われた検証と、新たに最適化された薬物の開発のための処理が続く。これまで、数式また?...

伝統的な漢方薬の研究と応用に関心が高まっています。化学成分が明確な現代薬とは対照的に、ハーブ処方には未知の成分があり、活性成分の送達を可能にするために抽出プロセスが必要です。多くの研究は、ハーブまたはハーブの処方全体の代表として、よく知られている構造を持つ1つの小さな化合物を選択しようとしていますが、薬理学的有効性やメカニズムは同等ではありません1,2 。フィンガープリンティングは複雑な漢方式の構成成分の分析を可能にするが、一部の成分は依然として明確に分析されていないため、研究3のすべての抽出物を組み合わせる際に問題が生じる。多数の成分/抽出物の相互作用は、漢方薬の治療効果を媒介する。この利点を保持するために、伝統的な抽出物の形態イオンは、依然として診療所で広く使用されています。抽出手順は治療効果に大きな影響を与えるため、特にインビボ研究では、伝統的な水煎出の標準プロトコールが必要である4,5。 他方、薬理学的機構を調べる場合、 インビトロまたはエクスビボの研究中の薬草煎じ薬の投与も課題である。薬物含有血清の概念は、1988年にTashinoによって最初に提案された6 。それ以来、数多くの研究者が漢方薬に適用しています7,8,9。薬物含有血清の方法は、血清の特定の成分の影響などのいくつかの制限があるが、依然として生理学的条件を厳密に模倣する方法であると考えられている。 14 、閉経骨粗鬆症15,16,17、早期卵巣不全18 、乳癌19 、卵巣癌20 、思春期遅発21の治療にも適用されています。ここでは、エラシアン煎剤（EXD）とその薬物含有血清の両方の調製のための詳細なプロトコールを提示する。加えて、我々は、ネズミ更年期骨粗鬆症モデルに対するEXDおよびEXD含有血清の適用を記載する。 EXDは、 9gのCurculigo orchioides Gaertn 、 Herba Epimedii </em&gt;、 Radix Morindae Officinalis 、 Radix Angelicae Sinensis 、 Cortex PhellodendriおよびRhizoma Anemarrhenaeそれぞれ6gを 1日当たりに投与する。マウスの等価線量は、以下の式22に基づいて0.1418EXD / kg /日である。dB = dA * RB / RA *（WA / WB） 1/3 。 dAおよびdBは、それぞれ、ヒトおよびマウスの体重当たりの用量（mg / kg）を示す。用量は、mg / kgで、EXD / kgの数で置き換えられる。 RAおよびRBは、（体表面積（m 2 ）/体重（kg）） 2/3 （ 表1参照）に比例するヒト身体因子およびマウス体係数をそれぞれ表す。 WAおよびWBは、それぞれヒトおよびマウスの体重（kg）を示す。

<table border="0" cellpadding="0" cellspacing="0" width="651">
	
	<tbody>
		<tr height="165">
			<td height="165" style="height:165px;width:216px;">Curculigo orchioides Gaertn (9&#8201;g), Herbaa Epimedii (9&#8201;g), Radix Morindae Officinalis (9&#8201;g), Radix Angelicae Sinensis (9&#8201;g), Cortex Phellodendri (6&#8201;g), and Rhizoma Anemarrhenae (6&#8201;g)</td>
			<td style="width:149px;">Kang-qiao Chinese Medicine Yinpian Co. Ltd (Shanghai, CN)</td>
			<td style="width:119px;">160922</td>
			<td style="width:167px;">EXD components</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Filter paper</td>
			<td style="width:149px;">GElifesciences</td>
			<td style="width:119px;">99-103-952</td>
			<td style="width:167px;">Filter EXD decoction before concentration</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Imprinting Control Region (ICR) mice</td>
			<td style="width:149px;">Shanghai Laboratory Animal Center</td>
			<td style="width:119px;">SCXK 2007-0005</td>
			<td style="width:167px;">In vivo study</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Sprague Dawley rats</td>
			<td style="width:149px;">Shanghai Laboratory Animal Center</td>
			<td style="width:119px;">SCXK 2007-0005</td>
			<td style="width:167px;">EXD-containing serum preparation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Syringe filter</td>
			<td style="width:149px;">Millipore</td>
			<td style="width:119px;">SLGP033RB</td>
			<td style="width:167px;">0.22 &#181;m</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">gavage needles (10 ml)</td>
			<td style="width:149px;">Shanghai BO Ge trade sales department</td>
			<td align="right" style="width:119px;">59104274</td>
			<td style="width:167px;">Adminstration of EXD</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Ketamine (80 mg/kg)&#160;</td>
			<td style="width:149px;">Fujian Gutian Pharma Co. Ltd</td>
			<td style="width:119px;">H35020148</td>
			<td>Anesthesia</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Xylazine (10 mg/kg)</td>
			<td style="width:149px;">Sunway Pharma Co. Ltd</td>
			<td style="width:119px;">CB07591</td>
			<td>Anesthesia</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:216px;">Dulbecco&#8217;s modification of Eagle&#8217;s medium Dulbecco (DMEM) culture medium</td>
			<td style="width:149px;">Gibco</td>
			<td style="width:119px;">12800-116</td>
			<td style="width:167px;">DMEM with 2 mM L-glutamine and without ribonucleosides and ribonucleotides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Streptomycin</td>
			<td style="width:149px;">Sigma</td>
			<td align="right" style="width:119px;">1277</td>
			<td style="width:167px;">100 &#181;g / ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin</td>
			<td style="width:149px;">Sigma</td>
			<td align="right" style="width:119px;">4687</td>
			<td style="width:167px;">100 &#181;g / ml</td>
		</tr>
	</tbody>
</table>

preparation of herbal medicine: er-xian decoction and er-xian-containing serum for in vivo and in vitro experiments

近年、臨床現場での代替薬である伝統的な漢方薬が注目されています。身体に送達する前に、生薬から活性成分を放出するためには、一般的に追加の抽出手順が必要である。水煎出は、臨床の場で広く使用されている古典的な抽出手順です。ここでは、薬草煎じ薬を実験研究に適用するために、エキソ煎じ薬（EXD）の詳細なプロトコルを提案する。 EXDの主要な4つのステップ（浸漬、水煎出、濾過、濃縮）と同様に、動物に適した投与量の計算が記載されています。さらに、血清含有EXDをインビトロでの検証の手段としてラットに導入する。ここでは、ラットにEXDを3日間経口投与した。次いで、血液サンプルを収集し、不活性化し、遠心分離し、濾過した。培養培地で希釈した血清を用いて、細胞または組織をvitro。例えば、EXDはインビボおよびインビトロ研究の両方に適用され、EXDが骨形成を増強することが実証された。このプロトコールは、漢方薬の調製および応用の参考として用いることができる。

OVXマウスの骨密度に対するEXDの効果腰椎切片のヘマトキシリンおよびエオシン（H＆E）染色は、OVX群（ 図1A 、左パネル）と比較して、 インビボでの EXD処置後の骨梁の増加を示す（ 図1A 、右パネル）。 図1Bは、OVXマウス（ 図1B 、左パネル）および12週間のEXD処置を行...

Watch this Scientific Journal Video about Preparation of Herbal Medicine: Er-Xian Decoction and Er-Xian-containing Serum for In Vivo and In Vitro Experiments at JoVE.com

Preparation of Herbal Medicine: Er-Xian Decoction and Er-Xian-containing Serum for In Vivo and In Vitro Experiments

ここでは、浸漬、煎出、ろ過、および濃縮の4段階でerxian decoction（EXD）の準備を行い、準備したEXD含有血清のラットへの投与を実証する。これらの方法は、伝統的な漢方薬などの薬草煎じ薬のin vivoおよびin vitro研究に適用可能である。

漢方薬の準備：Er-Xian煎じ薬とEr-Xian含有血清<em&gt;インビボ</em&gt;と<em&gt;インビトロ</em&gt;実験

Traditional herbal medicine, an alternative medicine in the clinical setting, has received increased attention in recent years. Before delivery to the ...

preparation-herbal-medicine-er-xian-decoction-er-xian-containing

Research

JoVE Journal

Medicine

Preparation of Herbal Medicine: Er-Xian Decoction and Er-Xian-containing Serum for In Vivo and In Vitro Experiments

10.9K ビュー.  Shanghai University of Traditional Chinese Medicine. ここでは、浸漬、煎出、ろ過、および濃縮の4段階でerxian decoction（EXD）の準備を行い、準備したEXD含有血清のラットへの投与を実証する。これらの方法は、伝統的な漢方薬などの薬草煎じ薬のin vivoおよびin vitro研究に適用可能である。 

ビデオ: Preparation of Herbal Medicine: Er-Xian Decoction and Er-Xian-containing Serum for In Vivo and In Vitro Experiments

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

細胞追跡のためのin vivoでの19 F MRI における

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

医学

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

マウスにおけるタキサンの薬物動態学的および薬力学的分析のための頸動脈煎茶

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same pathway, are likely to fail. Emphasis must be placed upon the inhibition of pathways that are responsible for intrinsic and/or adaptive resistance to therapy. An active field of investigation is the development and testing of DNA repair inhibitors that promote the action of, and prevent resistance to, commonly used chemotherapy and radiotherapy. We used a novel protocol to evaluate the effectiveness of BRCA2 inhibition as a means to sensitize tumor cells to the DNA damaging drug cisplatin. Tumor cell metabolism (acidification and respiration) was monitored in real-time for a period of 72 hr to delineate treatment effectiveness on a minute by minute basis. In combination, we performed an assessment of metastatic frequency using a chicken embryo chorioallantoic membrane (CAM) model of extravasation and invasion. This protocol addresses some of the weaknesses of commonly used in vitro and in vivo methods to evaluate novel cancer therapy regimens. It can be used in addition to common methods such as cell proliferation assays, cell death assays, and in vivo murine xenograft studies, to more closely discriminate amongst candidate targets and agents, and select only the most promising candidates for further development.

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Here, real-time monitoring of tumor cell metabolism, combined with an in vivo chicken embryo chorio-allantoic membrane (CAM) model of metastasis, are used to evaluate novel anti-cancer targets/agents for their ability to sensitize tumor cells to DNA damaging chemotherapeutics.

がん薬感の有効性を評価<em&gt;インビトロ</em&gt;と<em&gt;インビボ</em

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same ...

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.
First we describe how to generate subcutaneous tumors in mice, using antigen-negative cell lines as negative controls and antigen-positive cells as positive controls in the same mice for intraindividual comparison. We outline how to administer intravenously near-infrared fluorophore labelled (AlexaFluor680) antigen-specific nanobodies and conventional antibodies. In vivo imaging was performed with a small-animal NIRF-Imaging system. After the in vivo imaging experiments the mice were sacrificed. We then describe how to prepare the tumors for parallel ex vivo analyses by flow cytometry and fluorescence microscopy to validate in vivo imaging results.
The use of the near-infrared fluorophore labelled nanobodies allows for non-invasive same day imaging in vivo. Our protocols describe the ex vivo quantification of the specific labeling efficiency of tumor cells by flow cytometry and analysis of the distribution of the antibody constructs within the tumors by fluorescence microscopy. Using near-infrared fluorophore labelled probes allows for non-invasive, economical in vivo imaging with the unique ability to exploit the same probe without further secondary labelling for ex vivo validation experiments using flow cytometry and fluorescence microscopy.

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.

ナノボディ及び抗体ベースの検証<em&gt;インビボ</em使用してマウスにおける&gt;腫瘍異種移植NIRFイメージング実験<em&gt;エクスビボ</em&gt;フローサイトメトリーおよび顕微鏡

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

マウス収縮圧力を測定するために子宮内テレメトリ<em&gt;インビボ</em

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

ヒト腫瘍異種移植片からマウス細胞の枯渇は大幅に標的細胞の下流分析を向上させます

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic study in the liver. Here, numerous assays are described to comprehensively measure the phenotype and parameters of hepatic steatosis in mouse and hepatocyte models. 
Combining the physiological, histological, and biochemical methods, this system can be used to assess the progress of hepatic steatosis. In vivo, the measurements of body weight and nuclear magnetic resonance (NMR) provide a general understanding of mice in a non-invasive manner. Hematoxylin and Eosin (H&amp;E) and Oil Red O staining determine the histological morphology and lipid deposition of liver tissue under nutrient overload conditions, such as high-fat diet feeding. Next, the total lipid contents are isolated by chloroform/methanol extraction, which are followed by a biochemical analysis for triglyceride and cholesterol. Moreover, mouse primary hepatocytes are treated with high glucose plus insulin to stimulate lipid accumulation, an efficient in vitro model to mimic diet-induced hyperglycemia and hyperinsulinemia in vivo. Then, the lipid deposition is measured by Oil Red O staining and chloroform/methanol extraction. Oil Red O staining determines intuitive hepatic steatotic phenotypes, while the lipid extraction analysis determines the parameters that can be analyzed statistically. The present protocols are of interest to scientists in the fields of fatty liver diseases, insulin resistance, and type 2 diabetes.

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

の最適化分析<em&gt;インビボ</em&gt;と<em&gt;インビトロ</em&gt;脂肪肝

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

準備と<em&gt;インビトロ</em磁化のmiR修飾内皮細胞の&gt;キャラクタリゼーション

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

In VitroとIn Vivo検出、ひと細胞、線虫、マウス Mitophagy の

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

Preparation Of Gushukang (GSK) Granules for In Vivo and In Vitro Experiments

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang (GSK) granules, a marketed prescription in China, have bone-protective effects in treating POP. Before administration to the body, one standard preparation procedure is commonly required, which aims to promote the release of active constituents from raw herbs and enhance the pharmacological effects as well as therapeutic outcomes. This study proposes a detailed protocol for using GSK granules in in vivo and in vitro experimental assays. The authors first provide a detailed protocol to calculate the animal-appropriate dosages of granules for in vivo investigation: weighing, dissolving, storage, and administration. Second, this article describes protocols for micro-CT scanning and the measurement of bone parameters. Sample preparation, protocols for running the micro-CT machine and quantification of bone parameters were evaluated. Third, serum-containing GSK granules are prepared, and drug-containing serum is extracted for in vitro osteoclastogenesis and osteoblastogenesis. GSK granules were intragastrically administered twice per day to rats for three consecutive days. Blood was then collected, centrifuged, inactivated, and filtered. Finally, serum was diluted and used for performing osteoclastogenesis and osteoblastogenesis. The protocol described here can be considered a reference for pharmacological investigations of herbal prescription medicines, such as granules.

Preparation Of Gushukang GSK Granules for In Vivo and In Vitro Experiments

This article provides a detailed protocol for preparing a working solution of Gushukang granules for animal studies and GSK granule containing serum for in vitro experiments. This protocol can be applied to pharmacological investigations of herbal medicines as well as prescriptions for both in vivo and in vitro experiments.

インビボおよびインビトロ実験用グシュカン(GSK)顆粒の調製

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang ...