Ce travail a été soutenu par la National Natural Science Foundation of China (81573992). Nous remercions Emily K. Lo et Kathleen DiNapoli pour leur aide à l&#39;édition de langue.

Le protocole suit les directives sur les soins des animaux de l&#39;Université de médecine traditionnelle chinoise de Shanghai et est approuvé par l&#39;expérimentation animale du Comité d&#39;éthique animale de Shanghai. 1. Protocole I: Préparation de l&#39;EXD <ol><li> Calcul <ol><li> Administrer l&#39;EXD à un groupe de traitement de 10 souris de six mois de la région de contrôle d&#39;impression féminine (ICR) (0,02 kg / souris), 5 jours par semaine pendant 12 semaines. REMARQUE: un total de 1,702 EXDs est nécessaire. Étant donné que les EXD sont mesurés par incréments discrets, dans la pratique réelle, administrez 2 EXD. </li><li> Calculez le volume d&#39;EXD appliqué par souris ( c&#39;est-à-dire V = (0.1418 EXD / kg • jour) * (0.02 kg / souris) * 50 mL / EXD = 0.14 mL / mouse • day). </li></ol></li><li> Trempage <ol><li> Mettre toutes les matières premières de deux EXD ( c.-à-d., Curculigo orchioides Gaertn (18 g), Herbaa Epimedii (18 g), Radix Morindae Officinalis (18 g), RAdix Angelicae Sinensis (18 g), Cortex Phellodendri (12 g) et Rhizoma Anemarrhenae (12 g), 96 g au total) dans un récipient avec un couvercle. REMARQUE: Un récipient en céramique est recommandé. </li><li> Macer les herbes brutes avec 500 ml d&#39;eau distillée pendant 1 heure; L&#39;eau devrait couvrir les herbes d&#39;environ un pouce. Laissez toutes les herbes tremper soigneusement. </li></ol></li><li> Première décoction de l&#39;eau <ol><li> Chauffer les herbes à l&#39;aide d&#39;un cuisinière à gaz ou d&#39;une cuisinière à induction à haute puissance jusqu&#39;à ce que l&#39;eau bouillonne (environ 5 à 10 minutes, la durée dépend de la puissance de chauffage, du récipient et de la quantité d&#39;herbes et d&#39;eau). Mettez l&#39;appareil hors tension pendant 2 h. </li></ol></li><li> Première filtration <ol><li> Couvrir un gobelet en verre de 500 ml avec du papier filtre ordinaire ou avec de la gaze et du coton. Déposer soigneusement la décoction dans le bécher à travers le filtre. Laissez les herbes dans le récipient. </li><li> Renvoyer les résidus de fines herbesN le papier filtre sur le contenant. </li></ol></li><li> Deuxième décoction d&#39;eau <ol><li> Ajouter de l&#39;eau distillée au récipient avec les herbes; Laissez-le couvrir les herbes d&#39;environ un pouce. </li><li> Répétez l&#39;étape 1.3. </li></ol></li><li> Deuxième filtration <ol><li> Répétez les étapes 1.4.1 et 1.4.2. </li><li> Verser la deuxième décoction dans le même bécher. Mélanger ensemble la première et la seconde décoctions. </li></ol></li><li> Concentration <ol><li> Mettez le bécher sur un cuisinier à gaz avec de la gazine métallique sans amiante entre eux, faites chauffer la décoction à l&#39;aide d&#39;un mijot bas et remuez lentement et continuellement avec une tige de verre. </li><li> Lorsque la décoction se réduit à 200 mL, transférez-la à un bécher de 500 ml. </li><li> Répétez l&#39;étape 1.7.1 jusqu&#39;à ce que la décoction soit réduite à 100 mL. </li></ol></li><li> Stockage et administration <ol><li> Transférer la décoction concentrée dans une bouteille en verre stérile. Laissez refroidir à la température ambianteRe (RT). Conserver à 4 ° C si vous utilisez dans une semaine ou à -70 ° C pour un stockage à long terme. </li><li> À l&#39;aide d&#39;une aiguille de gavage, administrer 0,14 mL / souris de la décoction à des souris ovariectomisées (OVX) une fois par jour, 5 jours par semaine pendant 12 semaines. </li></ol></li></ol> 2. Protocole II: Préparation du Sérum contenant EXD <ol><li> Calcul <ol><li> Calculez la dose d&#39;équivalent de rat (ROUGE) en utilisant 0,15 kg comme poids corporel de chaque rat (1 mois): dB = (1/60) * (90/100) * (60 / 0.15) 1/3 = 0.111 EXD / Kg / jour. REMARQUE: Un total de 10 ml de sérum contenant EXD est nécessaire pour préparer 100 ml de milieu de culture (10% de sérum contenant EXD). Chaque rat devrait fournir 2 mL de sérum. Ainsi, 6 rats (une perte de 20% est pris en compte) sont nécessaires pour l&#39;administration EXD une fois par jour pendant 3 jours. Le nombre d&#39;EXD est: (0.111 EXD / kg • jour) * (0.15 kg / rat) * (6 rats) * (3 jours) = 0.300 EXD. Encore une fois, parce que les EXD sont mesurés par incréments discrets,Utilisez 1 EXD. </li><li> Calculer le volume d&#39;EXD appliqué par rat ( c.-à-d., V = (0.111 EXD / kg • jour) * (0.15 kg / rat) * (50 mL / EXD) = 0.83 mL / rat • jour). </li></ol></li><li> Trempage <ol><li> Mettez les matières premières pour 1 EXD dans un récipient avec un couvercle. Macerer les herbes brutes dans de l&#39;eau distillée pendant 1 heure; L&#39;eau devrait couvrir les herbes d&#39;environ un pouce. Laissez toutes les herbes tremper soigneusement. </li></ol></li><li> Première décoction de l&#39;eau <ol><li> Chauffer les herbes à l&#39;aide d&#39;une cuisinière à induction à haute puissance jusqu&#39;à ce que l&#39;eau bouillonne (environ 5 à 10 minutes, la durée dépend de la puissance de la chaleur, du récipient et de la quantité d&#39;herbes et d&#39;eau). Mettez l&#39;alimentation hors tension à feu doux pendant 2 h. </li></ol></li><li> Première filtration <ol><li> Couvrir un gobelet en verre de 500 ml avec du papier filtre ordinaire ou avec de la gaze et du coton. Déposer soigneusement la décoction dans le bécher à travers le filtre. Laissez les herbes dans le récipient. </li><li&gt; Retournez les résidus d&#39;herbes sur le papier filtre sur le récipient. </li></ol></li><li> Deuxième décoction d&#39;eau <ol><li> Ajouter de l&#39;eau distillée au récipient avec les herbes; Laissez-le couvrir les herbes d&#39;environ un pouce. </li><li> Répétez l&#39;étape 2.4. </li></ol></li><li> Deuxième filtration <ol><li> Répétez les étapes 2.5.1 et 2.5.2. </li><li> Verser la deuxième décoction dans le même bécher comme à l&#39;étape 2.4.1 et mélanger ensemble la première et la seconde décoctions. </li></ol></li><li> Concentration <ol><li> Mettez le bécher sur une cuisinière à gaz avec une gaze de fil sans amiante entre eux, faites chauffer la décoction à l&#39;aide d&#39;un mijot bas et remuez lentement et continuellement avec une tige en verre. </li><li> Lorsque la décoction se réduit à 100 mL, transférez-la à un bêcher de 100 ml. </li><li> Répétez l&#39;étape 2.7.1 jusqu&#39;à ce que la décoction se réduise à 50 mL. </li></ol></li><li> Stockage et administration <ol><li> Transférer la décoction concentrée en une stérileBouteille en verre. Laissez refroidir à la RT. Conserver à 4 ° C si vous utilisez dans une semaine ou à -70 ° C pour un stockage à long terme. </li><li> Administrer intragastriquement 0,83 ml / rat une fois par jour pendant 3 jours. </li></ol></li><li> Préparation de sérum contenant EXD <ol><li> Anesthésier les rats par injection intraperitoneale de 300 mL / 100 g de 80 mg / kg de kétamine et 10 mg / kg de xylazine à 1 h après la dernière administration d&#39;EXD. Confirmer une anesthésie appropriée par pincement. </li><li> Incitez la peau et le péritoine du rat de l&#39;abdomen au bas du thorax à l&#39;aide d&#39;un scalpel (ou des ciseaux à action directe). Créez une incision de longueur et de profondeur d&#39;environ 5 cm et 0,5 cm, respectivement. Déplacez les viscères abdominaux vers la gauche en utilisant du papier de soie. </li><li> À l&#39;aide de papier de soie, retirer le tissu conjonctif de l&#39;aorte abdominale pour exposer clairement le vaisseau. </li><li> Dessinez lentement le sang de l&#39;aorte abdominale en utilisant une seringue de calibre 22 de 10 ml. Transférer àUn tube stérile de 15 ml après avoir retiré l&#39;aiguille; Un rat peut produire 8 à 10 ml de sang. REMARQUE: Le rat doit être vivant ( c.-à-d. Avec l&#39;impulsion de l&#39;aorte abdominale) lorsque vous commencez le dessin du sang et que vous avez décroché après la fin du dessin. </li><li> Incorporer le sang dans une position verticale pendant 30 à 60 min à la RT de la pièce. Centrifuger à 500-600 xg pendant 20 min. Placez soigneusement tout le surnageant (sérum) dans un tube stérile de 50 ml et mélangez ensemble le sérum (de différents animaux). </li><li> Effectuer l&#39;inactivation de la chaleur en incubant le sérum dans un bain d&#39;eau à 56 ° C pendant 30 min. Filtrer le sérum en utilisant un filtre à seringue avec une membrane de polyéthersulfone hydrophile de taille de pores de 0,22 μm. Utiliser le sérum frais ou le conserver à -20 ° C. </li></ol></li><li> Préparation de sérum contenant un médicament témoin (utilisé dans le groupe témoin) <ol><li> Administrer chaque rat avec le même volume de solution saline (0,83 ml) une fois par jour pendant 3 jours. Les autres étapes sont identiques à celles de l&#39;étape 2.9.</li></ol></li><li> Application <ol><li> Ajouter 10 ml de sérum contenant un excès de SOR ou de médicament contenant EXD à chaque 100 ml de milieu, plus 1% de pénicilline-streptomycine (PS) 23 . </li></ol></li></ol>

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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>

Les auteurs déclarent qu&#39;ils n&#39;ont pas d&#39;intérêts financiers concurrents.

Au cours des dernières années, on a accordé plus d&#39;attention aux médicaments à base de plantes, un type de médicament alternatif appliqué dans le milieu clinique dans le monde oriental depuis des milliers d&#39;années. Différente du modèle de la médecine moderne &quot;banc à lit&quot;, la médecine traditionnelle à base de plantes nécessite d&#39;abord le modèle &quot;lit à banc&quot; pour expliquer leurs mécanismes. Cela peut être suivi d&#39;une validation effectuée au niveau du banc et du trait...

L&#39;intérêt pour l&#39;étude et l&#39;application de la phytothérapie traditionnelle augmente actuellement. Par opposition aux médicaments modernes, dans lesquels les ingrédients chimiques sont définis, les formules à base de plantes contiennent des ingrédients inconnus et nécessitent des procédés d&#39;extraction pour permettre la distribution de leurs composés actifs. Bien que de nombreuses études tentent de sélectionner un petit composé avec une structure bien connue comme représentant de l&#39;herbe entière ou de la formule à base de plantes, ni les efficacités pharmacologiques ni les mécanismes ne peuvent être considérés comme équivalents 1 , 2 . Bien que les empreintes digitales permettent d&#39;analyser les constituants des formules à base de plantes complexes, certains constituants ne sont toujours pas clairement analysés, ce qui entraîne un défi lors de la combinaison de tous les extraits d&#39;étude 3 . Les interactions de nombreux constituants / extraits mesurent les effets thérapeutiques des plantes médicinales. Pour conserver cet avantage, la forme-décoct traditionnelle d&#39;extraitIon - est encore largement utilisé dans la clinique. Comme la procédure d&#39;extraction a un impact important sur l&#39;efficacité thérapeutique, un protocole standard de décoction traditionnelle d&#39;eau est nécessaire, en particulier pour les études in vivo 4 , 5 . D&#39;autre part, lors de l&#39;exploration de mécanismes pharmacologiques, l&#39;administration de décoctions à base de plantes lors d&#39;études in vitro ou ex vivo est également un défi. La notion de sérum contenant des médicaments a d&#39;abord été proposée par Tashino en 1988 6 . Depuis lors, un nombre croissant de chercheurs l&#39;ont appliquée à la phytothérapie 7 , 8 , 9 . Bien que la méthode du sérum contenant du médicament présente certaines limites, telles que l&#39;influence de certains composants du sérum, elle est encore considérée comme une méthode qui imite de près les conditions physiologiques. 10 , 11 , 12 , 13 . Il a également été appliqué au traitement de l&#39;anémie aplasique 14 , l&#39;ostéoporose ménopausique 15 , 16 , 17 , l&#39;insuffisance ovarienne prématurée 18 , le cancer du sein 19 , le cancer de l&#39;ovaire 20 et la puberté retardée 21 . Ici, nous présentons des protocoles détaillés pour la préparation à la fois de la décoction er-xian (EXD) et de son sérum contenant des médicaments. De plus, nous décrivons l&#39;application de sérum contenant EXD et EXD aux modèles d&#39;ostéoporose ménopausique murine. Un EXD est composé de 9 g chacun de Curculigo orchioides Gaertn , Herbaa Epimedii </eM&gt;, Radix Morindae Officinalis et Radix Angelicae Sinensis et 6 g de Cortex Phellodendri et Rhizoma Anemarrhenae par patient adulte par jour. La dose équivalente pour une souris est de 0,1418 EXD / kg / jour selon l&#39;équation suivante 22 : dB = dA * RB / RA * (WA / WB) 1/3 . DA et dB font référence à la dose par poids corporel (mg / kg) de l&#39;homme et de la souris, respectivement. La dose, en mg / kg, est remplacée par le nombre d&#39;EXD / kg. La RA et la RB représentent respectivement le facteur corporel humain et le facteur corporel de la souris, qui sont proportionnels à la (surface corporelle (m 2 ) / poids corporel (kg)) 2/3 (voir tableau 1 ). WA et WB indiquent le poids corporel (kg) de l&#39;homme et de la souris, respectivement.

<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

La médecine traditionnelle à base de plantes, une médecine alternative dans le milieu clinique, a reçu une attention accrue ces dernières années. Avant la livraison au corps, une procédure d&#39;extraction supplémentaire est communément requise pour libérer les constituants actifs des herbes brutes. La décoction de l&#39;eau est une procédure d&#39;extraction classique qui est encore largement utilisée dans les milieux cliniques. Ici, nous proposons un protocole détaillé pour la décoction er-xian (EXD) afin d&#39;appliquer des décoctions à base de plantes à des études expérimentales. On décrit le calcul d&#39;une dose adaptée aux animaux, ainsi que les quatre étapes principales de l&#39;EXD: trempage, décoction de l&#39;eau, filtration et concentration. De plus, l&#39;EXD contenant du sérum est introduit chez les rats comme moyen de validation in vitro . Ici, les rats ont été administrés par voie orale EXD pendant trois jours. Les échantillons de sang ont ensuite été recueillis, inactivés, centrifugés et filtrés. Le sérum, dilué avec le milieu de culture, peut être utilisé pour traiter les cellules ou les tissus dans vItro. Par exemple, EXD a été appliqué à la fois à des études in vivo et in vitro et a démontré que EXD améliore l&#39;ostéogenèse. Ce protocole peut être utilisé comme référence pour la préparation et l&#39;application de plantes médicinales.

L&#39;effet d&#39;EXD sur la densité osseuse des souris OVX La coloration de l&#39;hématoxyline et de l&#39;éosine (H &amp; E) de la colonne vertébrale lombaire montre des trabécules osseuses accrues après traitement EXD in vivo ( figure 1A , panneau droit) par rapport à celles du groupe OVX ( figure 1A , panneau de gauche). La figure 1B montre les images μCT rep...

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

Ici, nous présentons la préparation d&#39;une décoction er-xian (EXD) en quatre étapes, trempage, décoction, filtration et concentration, et démontrons l&#39;administration d&#39;un sérum contenant EXD préparé à des rats. Ces méthodes s&#39;appliquent à l&#39;étude in vivo et in vitro des décoctions à base de plantes telles que les médicaments traditionnels chinois.

Préparation de la phytothérapie: la décoction Er-Xian et le sérum contenant de l&#39;er-Xian pour<em&gt; In vivo</em&gt; Et<em&gt; In Vitro</em&gt; Expériences

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

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Medicine

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

10.9K vues.  Shanghai University of Traditional Chinese Medicine.  Ici, nous présentons la préparation d'une décoction er-xian (EXD) en quatre étapes, trempage, décoction, filtration et concentration, et démontrons l'administration d'un sérum contenant EXD préparé à des rats. Ces méthodes s'appliquent à l'étude in vivo et in vitro des décoctions à base de plantes telles que les médicaments traditionnels chinois. 

Vidéo: 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.

In vivo 19 F IRM pour le suivi cellulaire

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. ...

Recherche

Médecine

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.

Infusions de l&#39;artère carotide pour pharmacocinétiques et pharmacodynamiques Analyse des taxanes chez la souris

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.

Évaluation de l&#39;efficacité des médicaments contre le cancer Sensibilisation<em&gt; In Vitro</em&gt; Et<em&gt; In Vivo</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.

Validation des Nanobodies et Anticorps Based<em&gt; In Vivo</em&gt; Expériences tumeur xénogreffe NIRF-imagerie dans des souris en utilisant<em&gt; Ex Vivo</em&gt; Cytométrie en flux et microscopie

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.

Télémétrie intra-utérine mesurer souris contractile pression<em&gt; In Vivo</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.

Appauvrissement des cellules de souris de xénogreffes de tumeurs humaines améliore de manière significative l&#39;analyse en aval des cellules cibles

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.

Analyse optimisée des<em&gt; In Vivo</em&gt; et<em&gt; In vitro</em&gt; Stéatose hépatique

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.

préparation et<em&gt; In vitro</em&gt; Caractérisation des aimanté miR-modifié cellules endothéliales

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 et In Vivo la détection des Mitophagy dans les cellules humaines, C. Eleganset la souris

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.

Préparation des granules de Gushukang (GSK) pour les expériences in vivo et in vitro

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

Préparation de la phytothérapie: la décoction Er-Xian et le sérum contenant de l'er-Xian pour

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In Vitro et In Vivo la détection des Mitophagy dans les cellules humaines, C. Eleganset la souris

Préparation des granules de Gushukang (GSK) pour les expériences in vivo et in vitro