Bu çalışma Çin Ulusal Doğa Bilimleri Vakfı (81573992) tarafından desteklenmiştir. Dil düzenleme yardımı için Emily K. Lo ve Kathleen DiNapoli&#39;ye teşekkür ediyoruz.

Protokol, Şanghay Geleneksel Çin Tıbbı&#39;nın hayvan bakım rehberlerini takip eder ve hayvan deneyleri Şanghay Hayvan Etik Komitesi tarafından onaylanır. 1. Protokol I: EXD&#39;nin hazırlanması <ol><li> Hesaplama <ol><li> EXD&#39;yi 10 altı aylık bayan Baskı Kontrol Bölgesi (ICR) farelerine (0.02 kg / fare), haftada 5 gün 12 hafta süreyle uygulayan bir tedavi grubuna uygulayın. NOT: Toplam 1.702 EXD&#39;ye ihtiyaç vardır. EXD&#39;ler ayrı artışlarla ölçülür, çünkü gerçek uygulamada 2 EXD uygulayın. </li><li> Fare başına uygulanan EXD hacmini hesaplayın ( yani, V = (0.1418 EXD / kg • gün) * (0.02 kg / fare) * 50 mL / EXD = 0.14 mL / fare • gün. </li></ol></li><li> Islatma <ol><li> İki EXD&#39;nin ( örneğin Curculigo orchioides Gaertn (18 g), Herbaa Epimedi (18 g), Radix Morindae Officinalis&#39;in (18 g), RAdix Angelicae Sinensis (18 g), Cortex Phellodendri (12 g) ve Rhizoma Anemarrhenae (12 g), toplam 96 g) bir kapaklı bir kaba koydu. NOT: Seramik bir kap tavsiye edilir. </li><li> Çiğ otları 500 mL damıtılmış su ile 1 saat süreyle macerante edin; Su otları bir inç kadar kaplamalıdır. Tüm otları iyice ıslatın. </li></ol></li><li> İlk suda kaynatma <ol><li> Otlar, su kaynarcaya kadar yüksek sıcaklıkta bir gaz ocağı veya bir indüksiyon ocağı kullanarak ısıtın (yaklaşık 5 - 10 dakika, süre, ısıtma gücüne, kaba ve otların miktarı ve suya bağlıdır). Gücü 2 saat daha düşük bir kaynatma değere çevirin. </li></ol></li><li> İlk filtrasyon <ol><li> Normal filtre kağıdıyla veya gazlı bezle ve pamuklu, 500 mL&#39;lik cam bir behere koyun. Dikkatli bir şekilde, kaynatmayı filtre vasıtasıyla behergöre dökün. Şifalı bitkiler kabın içinde bırakın. </li><li> Geri kalan bitki kalıntılarını geri getirin oFiltre kağıdını kaba koyun. </li></ol></li><li> İkinci suda kaynatma <ol><li> Otlarla birlikte kaba damıtılmış su ekleyin; Otları bir inç kadar kapağı olsun. </li><li> Adım 1.3&#39;ü tekrarlayın. </li></ol></li><li> İkinci filtrasyon <ol><li> 1.4.1 ve 1.4.2 adımlarını tekrarlayın. </li><li> İkinci kaynatmayı aynı behere dökün. Birinci ve ikinci dekatikleri birlikte karıştırın. </li></ol></li><li> konsantrasyon <ol><li> Beferleri asbest içermeyen tel gazlı bezli bir gaz ocağının üzerine koyun, kaynatıp düşük bir kaynatmayla ısıtın ve yavaşça ve bir cam çubukla karıştırın. </li><li> Yoğuşma 200 mL&#39;ye düştüğünde, 500 mL&#39;lik bir behere aktarın. </li><li> Yıkama 100 mL&#39;ye düşene kadar adım 1.7.1&#39;i tekrarlayın. </li></ol></li><li> Depolama ve yönetim <ol><li> Konsantre kaynatmayı steril bir cam şişeye aktarın. Oda sıcaklığına kadar soğutunYeniden (RT). Bir hafta içinde kullanıyorsanız 4 ° C&#39;de veya uzun süre depolamada -70 ° C&#39;de saklayın. </li><li> Bir tenteli iğne kullanarak, ovarektomize (OVX) farelere günde bir kez, 12 hafta boyunca haftanın 5 günü, 0.14 mL / fare eksiltme uygulayın. </li></ol></li></ol> 2. Protokol II: EXD-içeren Serumun Hazırlanması <ol><li> Hesaplama <ol><li> Her sıçanın vücut ağırlığı (1 aylık) olarak 0.15 kg kullanarak sıçan eşdeğer dozunu (RED) hesaplayın: dB = (1/60) * (90/100) * (60 / 0.15) 1/3 = 0.111 EXD / kg / gün. NOT: 100 mL kültür ortamı (% 10 EXD içeren serum) hazırlamak için toplam 10 mL EXD içeren serum gereklidir. Her sıçandan 2 mL serum sağlaması bekleniyor. Böylece, 6 sıçan (% 20&#39;lik bir kayıp dikkate alınır), 3 gün boyunca günde bir kez EXD uygulaması için gereklidir. EXD sayısı: (0.111 EXD / kg • gün) * (0.15 kg / sıçan) * (6 sıçan) * (3 gün) = 0.300 EXD. Yine, EXD&#39;ler ayrı artışlarla ölçülür,1 EXD kullanın. </li><li> Sıçan başına uygulanan EXD hacmini hesaplayın ( yani, V = (0.111 EXD / kg • gün) * (0.15 kg / sıçan) * (50 mL / EXD) = 0.83 mL / sıçan • gün. </li></ol></li><li> Islatma <ol><li> 1 EXD&#39;ye ait hammaddeleri kapaklı bir kaba koyun. Çiğ otları damıtılmış suda 1 saat maceratlayın; Su otları bir inç kadar kaplamalıdır. Tüm otları iyice ıslatın. </li></ol></li><li> İlk suda kaynatma <ol><li> Otlar, yüksek kaynaklarda kaynar su gelene kadar indüksiyon ocağı kullanarak ısıtın (yaklaşık 5 - 10 dakika; süre, ısı gücüne, konteynere ve otlar ile suyun miktarına bağlıdır). Gücü 2 saat daha düşük bir kaynatma noktasına getirin. </li></ol></li><li> İlk filtrasyon <ol><li> Normal filtre kağıdıyla veya gazlı bezle ve pamuklu, 500 mL&#39;lik cam bir behere koyun. Dikkatli bir şekilde, kaynatmayı filtre vasıtasıyla behergöre dökün. Şifalı bitkiler kabın içinde bırakın. </li><li&gt; Filtre kağıdındaki bitki kalıntılarını kaba geri koyun. </li></ol></li><li> İkinci suda kaynatma <ol><li> Otlarla birlikte kaba damıtılmış su ekleyin; Otları bir inç kadar kapağı olsun. </li><li> Adım 2.4&#39;ü tekrarlayın. </li></ol></li><li> İkinci filtrasyon <ol><li> 2.5.1 ve 2.5.2 adımlarını tekrarlayın. </li><li> İkinci sıkma işlemini, adım 2.4.1&#39;deki gibi aynı behça boşaltın ve birinci ve ikinci dekatikleri birlikte karıştırın. </li></ol></li><li> konsantrasyon <ol><li> Asfalt içermeyen tel gazlı bezli bir gaz ocağı üzerine beheri koyun, kaynatmayı düşük bir kaynatmayla ısıtın ve yavaşça ve bir cam çubukla karıştırın. </li><li> Yoğuşma 100 mL&#39;ye düştüğünde, 100 mL&#39;lik bir behere aktarın. </li><li> Yıkama 50 mL&#39;ye düşene kadar adım 2.7.1&#39;i tekrarlayın. </li></ol></li><li> Depolama ve yönetim <ol><li> Konsantre kaynatmayı sterilizeIle cam şişe. Oda sıcaklığına soğumasını bekleyin. Bir hafta içinde kullanıyorsanız 4 ° C&#39;de veya uzun süre depolamada -70 ° C&#39;de saklayın. </li><li> Intragastional olarak 0.83 mL / sıçan günde bir kez 3 gün boyunca uygulayın. </li></ol></li><li> EXD içeren serum preparatı <ol><li> Farelerin son uygulanmasından 1 saat sonra intraperitonal olarak 300 mL / 100 g 80 mg / kg ketamin ve 10 mg / kg xylazin enjekte ederek anestezi uygulayın. Çimdikleme ile doğru anestezi doğrulayın. </li><li> Sıçanın cildi ve peritonunu, karın bölgesinden göğüs dibine bir bistül (veya düz ameliyat makası) kullanarak incise edin. Sırasıyla yaklaşık 5 cm ve 0.5 cm&#39;lik bir uzunluk ve derinlik kesiği oluşturun. Doku kağıdı kullanarak karın iç organını sola doğru hareket ettirin. </li><li> Doku kağıdı kullanarak, damarı açıkça ortaya çıkarmak için karın aortunun bağ dokusunu çıkarın. </li><li> Karbon aortundan, 10 mL&#39;lik, 22 ölçüslü bir şırınga kullanarak kanı yavaşça çekin. Şuraya aktar:İğneyi çıkardıktan sonra 15 mL&#39;lik steril bir tüp; Bir fare 8-10 mL kan üretebilir. NOT: Kan çizimine başlanırken ve çizim tamamlandıktan sonra ölü olan sıçan canlı olmalıdır ( yani, abdominal aort titreşirken). </li><li> Oda RT&#39;da 30 - 60 dakika boyunca dik bir konumda kan pıhtılaştırın. 500-600 xg&#39;de 20 dakika boyunca santrifüjleyin. Tüm süpernatanı (serum) dikkatli bir şekilde bir 50 mL steril tüp içine yerleştirin ve tüm serumu (farklı hayvanlardan) karıştırın. </li><li> Serumu 56 ° C&#39;lik bir su banyosunda 30 dakika inkübe ederek ısı inaktivasyonu yapın. Serumu, 0.22 um gözenek boyutlu bir hidrofil polietersülfon membranlı bir şırınga filtresi kullanarak filtreleyin. Serumu taze veya -20 ° C&#39;de saklayın. </li></ol></li><li> Kontrollü ilaç içeren serum preparatı (kontrol grubunda kullanılır) <ol><li> Her bir fare günde bir kez aynı miktarda tuzlu su (0.83 mL) ile 3 gün süre ile idare edilir. Diğer adımlar adım 2.9&#39;daki ile aynıdır.</li></ol></li><li> Uygulama <ol><li> Her 100 ml&#39;lik orta ve artı% 1 penisilin-streptomisin (PS) 23&#39;e 10 mL EXD içeren serum veya ilaç içeren serum ekleyin. </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>

Yazarlar, rekabet eden mali çıkarlarının olmadığını beyan ettiler.

Son yıllarda binlerce yıldır Doğu dünyasında klinik ortamda uygulanan alternatif tıp türlerinden biri olan bitkisel ilaçlara daha fazla önem verilmektedir. Modern tıbbın &quot;tezgahdan tezgah&quot; deseninden farklı olarak, geleneksel bitkisel tıbbın ilk önce kendi mekanizmalarını açıklamak için &quot;tezgah yatağı&quot; örneğini gerektirir. Bunu, tezgah aşamasında yapılan doğrulama ve yeni optimize edilmiş ilaçların geliştirilmesi için işleme takip edilebilir. Şu ana kadar, bütün ...

Çalışmaya ve geleneksel bitkisel tıbbın uygulanmasına olan ilgi günümüzde artmaktadır. Kimyasal maddelerin kesin olduğu modern ilaçların aksine bitkisel formüllerin bilinmeyen bazı bileşenleri vardır ve aktif bileşiklerinin verilmesini sağlamak için ekstraksiyon işlemleri gerektirir. Çoğu araştırma, bütün ot veya bitkisel formülün temsilcisi olarak iyi bilinen bir yapıya sahip küçük bir bileşik seçmeye çalışsa da, ne farmakolojik etkinlikler ne de mekanizmalar eşdeğer 1 , 2 olarak kabul edilemez. Parmak izi kompleks bitkisel formüllerin bileşenlerini analiz etmeyi sağlarken, bazı bileşenler hala net bir şekilde analiz edilmediğinden, çalışma 3 için tüm özütleri birleştirirken bir meydan okumaya neden olur. Çok sayıda bileşenin / ekstraktların etkileşimleri, bitkisel ilaçların terapötik etkilerine aracılık eder. Bu avantajı korumak için, geleneksel ekstrakt form-dekoctİyon hala klinikte yaygın olarak kullanılmaktadır. Ekstrakt prosedürü, terapötik etkinlik üzerinde büyük bir etkiye sahip olduğu için, özellikle in vivo çalışmalar için 4 , 5 için geleneksel su sıkılmasının standart bir protokolü gereklidir. Öte yandan, farmakolojik mekanizmaları araştırırken, in vitro veya ex vivo çalışmalar sırasında bitkisel açma maddelerinin uygulanması da bir mücadeledir. İlaç içeren serumun konsepti ilk kez Tashino tarafından 1988&#39;de 6 önerildi. O zamandan beri, artan sayıda araştırmacı onu bitkisel ilaç 7 , 8 , 9&#39;a uyguladı. İlaç içeren serumun metodu, serumun belli bileşenlerinin etkisi gibi bazı kısıtlamalara sahip olmakla birlikte, fizyolojik koşulları yakından taklit eden bir yöntem olarak kabul edilmektedir. 14 , menopoz osteoporozu 15 , 16 , 17 , prematür over yetmezliği 18 , meme kanseri 19 , yumurtalık kanseri 20 ve ertelenmiş ergenlik 21&#39;in tedavisinde de uygulanmıştır. Burada, er-xian ekstraksiyonunun (EXD) ve ilacı içeren serumun hazırlanması için ayrıntılı protokoller sunuyoruz. Buna ek olarak, fare menopoz osteoporotik modellerine EXD ve EXD içeren serumun uygulanmasını açıklıyoruz. Bir EXD, 9 gr Curculigo orchioides Gaertn , Herbaa Epimedii&#39;den oluşur </eM&gt;, Radix Morindae Officinalis ve Radix Angelicae Sinensis ve her biri yetişkin hasta başına günde 6 g Cortex Phellodendri ve Rhizoma Anemarrhenae . Bir fare için eşdeğer doz, aşağıdaki denklem 22&#39;ye dayanılarak 0.1418 EXD / kg / gün&#39;dür: dB = dA * RB / RA * (WA / WB) 1/3 . DA ve dB, sırasıyla insan ve fare vücut ağırlığı başına doz (mg / kg) anlamına gelir. Dozun mg / kg olarak, EXD / kg sayısı ile değiştirilir. RA ve RB sırasıyla vücut yüzey alanı (m 2 ) / vücut ağırlığı (kg) ile orantılı insan vücut faktörünü ve fare gövde faktörünü temsil eder) 2/3 (Bkz. Tablo 1 ). WA ve WB sırasıyla insan ve fare vücut ağırlığını (kg) gösterir.

<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

Klinik ortamda alternatif bir ilaç olan geleneksel bitki tıbbı, son yıllarda artan bir ilgi görmüştür. Vücutta teslim edilmeden önce, aktif çiğneme maddelerini çiğ otlardan ayırmak için ek bir ekstraksiyon prosedürü yaygın olarak gereklidir. Su dekompozisyonu, klinik ortamda halen yaygın olarak kullanılan klasik bir ekstraksiyon prosedürüdür. Burada, deneysel araştırmalara bitki özütleri uygulamak için er-xian kaynatma işlemi (EXD) için ayrıntılı bir protokol önermekteyiz. Hayvana uygun doz hesaplamanın yanı sıra EXD&#39;nin dört ana aşamasını açıklar: ıslatma, su dekompozisyonu, filtrasyon ve konsantrasyon. Buna ek olarak, serum içeren EXD, in vitro doğrulama aracı olarak farelere verilir. Burada sıçanlar oral yoldan üç gün süreyle EXD ile tedavi edildi. Daha sonra kan numuneleri toplandı, inaktive edildi, santrifüj edildi ve filtrelendi. Kültür ortamı ile seyreltilmiş serum, v&#39;de hücreleri veya dokuları tedavi etmek için kullanılabilirItro. Örneğin, EXD, hem in vivo hem de in vitro çalışmalarda uygulanmış ve EXD&#39;nin osteojenezi arttırdığını göstermiştir. Bu protokol bitkisel ilaçların hazırlanması ve uygulanması için referans olarak kullanılabilir.

EXD&#39;nin OVX farelerinin kemik yoğunluğuna etkisi Omurga vertebrası bölümündeki hematoksilin ve eozin boyama, OVX grubundaki kişilerle ( Şekil 1A , sol panel) karşılaştırıldığında in vivo EXD tedavisinden sonra artmış kemik trabekülleri gösterir ( Şekil 1A , sağ panel). Şekil 1B , 12 haftalık EXD tedavisine sahip OVX farelerindeki ( Şe...

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

Burada, erüe, ekstraksiyon, filtrasyon ve konsantrasyon olmak üzere dört basamakta bir er-xian ekstraksiyonunun (EXD) hazırlanması ve hazırlanmış bir EXD içeren serumun sıçanlara uygulanmasını gösteriyoruz. Bu yöntemler, geleneksel Çin ilaçları gibi bitkisel dekompozisyonların in vivo ve in vitro çalışması için geçerlidir.

Bitkisel Tıbbın Hazırlanması: Er-Xian Kaynatma ve Er-Xian içeren Serum<emIn vivo</emVe<em&gt; In vitro</em&gt; Deneyler

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

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JoVE Journal

Medicine

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

10.9K Görüntüleme.  Shanghai University of Traditional Chinese Medicine.  Burada, erüe, ekstraksiyon, filtrasyon ve konsantrasyon olmak üzere dört basamakta bir er-xian ekstraksiyonunun (EXD) hazırlanması ve hazırlanmış bir EXD içeren serumun sıçanlara uygulanmasını gösteriyoruz. Bu yöntemler, geleneksel Çin ilaçları gibi bitkisel dekompozisyonların in vivo ve in vitro çalışması için geçerlidir. 

Video: 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.

Hücre Takip için vivo 19 F MRG

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. ...

Tıp

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.

Farelerde Taksanlar Farmakokinetik ve Farmakodinamik Analiz Karotis Arter İnfüzyonlar

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.

Kanser İlaç Duyarlılık Etkinliğinin Değerlendirilmesi<em&gt; İn Vitro</em&gt; Ve<em&gt; İn 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.

Nanobody ve Antikor Temelli Doğrulama<em&gt; İn Vivo</emFarelerde&gt; Tümör Xenograftlarında NIRF-görüntüleme Deneyler kullanma<em&gt; Ex vivo</em&gt; Sitometrisi ve Mikroskopi

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.

Rahim içi Telemetri Fare Kontraktil Basıncını Ölçmek<em&gt; İn 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.

İnsan Tümör ksenogratflarının gelen Fare Hücrelerinin tükenmesi Anlamlı Hedef Hücre Alt Analizi geliştirir

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.

Optimize Analizi<em&gt; İn Vivo</em&gt; ve<em&gt; İn Vitro</em&gt; Karaciğer Steatoz

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.

Hazırlık ve<em&gt; İn Vitro</em&gt; Mıknatıslanmış karakterizasyonu Endotel Hücreleri miR-modifiye

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.

Vitro ve In Vivo insan hücreleri, C. Elegansve fareler Mitophagy tespiti

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.

In vivo ve In vitro deneyler için Gushukang (GSK) granül hazırlanması

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

Bitkisel Tıbbın Hazırlanması: Er-Xian Kaynatma ve Er-Xian içeren Serum

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