Name: preparation of gushukang (gsk) granules for in vivo and in vitro experiments
Uploaded: 2019-05-09T12:30:00
Description: Watch this Scientific Journal Video about Preparation Of Gushukang GSK Granules for In Vivo and In Vitro Experiments at JoVE.com

这项研究得到了中国国家自然科学基金(81804116、81673991、81770107、81603643和81330085)的资助,该项目由中国科技部创新团队项目(2015RA4002至WYJ)资助。中国教育部创新团队(IRT1270至WYJ)、上海中医慢性病医学中心(2017ZZ01010至WYJ)、《三年行动加快中医药发展计划》(ZY(ZY(2018-2020)-CCCX-3003至WYJ)和国家重点研究开发项目(2018YFC1704302)。

所有实验程序均经上海中医药大学动物护理与使用委员会(SZY201604005)批准。
1. 葛兰素史克工作解决方案的准备和管理
<ol><li>计算小鼠的葛兰素史克颗粒的等效剂量。<ol>
<li>基于 Meeh-Rubner 方程15计算身体表面 :身体表面 = K x (体重2/3) /1000,其中 K 值为 10.6,用于人类,9.1 表示鼠标。假设人体重量为70公斤,则人体表面(m 2)= 10.6 x (702/3)/1000 = 1.8 m2</sup...

<ol>
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L., Wang, L., Bi, X. L., Chen, B. B., 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=Gushukang+exerts+osteopreserve+effects+by+regulating+Vitamin+D+and+Calcium+metabolism+in+ovariectomized+mice.">Gushukang exerts osteopreserve effects by regulating Vitamin D and Calcium metabolism in ovariectomized mice.</a> Journal of Bone Mineral Metabolism. , 1-11 (2018).</li><li>Cui, S. Q., 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=Mechanistic+study+of+Shen+(Kidney)tonifying+prescription+Gushukang+in+Preventing+and+Treating+Primary+Osteoporosis.">Mechanistic study of Shen (Kidney)tonifying prescription Gushukang in Preventing and Treating Primary Osteoporosis.</a> Journal of Chinese Medical University. 30 (16), 351-354 (2001).</li><li>Wang, Y., Shang, K., Li, Y. K., Tao, X. 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中药制剂的颗粒已成为配方或处方的常见选择之一。葛兰素史克颗粒由几种基于临床经验或中医理论的草药组成,具有较好的疗效,副作用少4。与水解相比,颗粒具有以下优点:口感好、交货方便、长期储存、标准方案、疗效一致、生产效率高。目前,颗粒是中医中最常用的药学形态之一。然而,药理作用的基本机制仍然很少被研究。有必要确定制备颗粒的关键步骤,以研究潜在的药理?...

中医是治疗骨质疏松症的重要补充和替代方法之一。水净化是公式3的基本和最常用的形式。然而,缺点也存在:味道不好,运输不便,保质期短,协议不一致,限制了使用以及治疗效果。为了避免上述缺点,以及追求更好的效果,颗粒被开发,并已被广泛应用4。虽然许多研究已经探索了一个或多个有效成分的药理机制从颗粒5,6,7,确切的机制和潜在的药理过程仍然是难以识别。这是因为一个颗粒中过多的有效成分可能同时产生类似或相反的效果4。因此,开发一个标准方案,在输送到人体之前制备颗粒,不仅对治疗效果有很大的影响,而且对体内和体外检测也是必需的。
此外,在临床颗粒的疗效难以确认和准确识别使用体外或体外研究,这带来了挑战,因为药理机制过于复杂。为了解决这个问题,Tashino于20世纪80年代首次提出制备含药物血清。从那时起,许多研究人员将含药物血清应用于草药,包括颗粒9,10,11。目前,选择含药物血清进行体外调查被认为是一种与生理条件密切相关的策略。
根据中医理论,在临床实践的基础上,研制出用于治疗绝经后骨质疏松症(POP)的古树康(GSK)颗粒。葛兰素史克颗粒可防止体内卵泡(OVX)小鼠的骨质流失,抑制骨细胞骨吸收,刺激成骨骨形成4。因此,Li等人12日发现,GSK颗粒通过增强钙受体的活性来刺激骨骼形成,对OVX小鼠具有骨保护作用。为了确认GSK颗粒的骨保护作用和药理作用,作者在这里提供了一个详细的程序,用于制备工作溶液和含有药物(GSK颗粒)的血清。此外,本文还介绍了GSK颗粒在OVX诱导的骨化小鼠模型和GSK含颗粒血清中的应用,用于体外骨细胞形成/骨细胞发生。
GSK颗粒由几种草药13、14组成,很容易完全溶解在盐水中。因此,盐水作为车辆。沙姆操作小鼠(Sham)和OVX小鼠施用的盐水量与颗粒施用小鼠相同。根据Meeh-Rubner方程15计算了小鼠的葛兰素史克颗粒的等效剂量。该方程不仅具有获得安全剂量的优点,而且保证了药理作用15。葛兰素史克颗粒的三种剂量如下: (1) GSKL: OVX = 低剂量葛兰素史克颗粒, 2 g/kg/天.(2) GSKM:OVX = 中剂量葛兰素史克颗粒,4 克/千克/天。(3) GSKH:OVX = 高剂量葛兰素史克颗粒,8克/千克/天。GSKL、GSKM和GSKH组的小鼠是胃内管理的葛兰素史克颗粒。碳酸钙(600毫克/片)与维生素D3(125国际单位/片剂),例如,在成熟和上市的产品(例如,钙酸盐[CAL])用于治疗和预防骨质疏松症,被用作积极的控制。

<table>
	<tbody>
		<tr>
			<td>&#945;-MEM</td>
			<td>Hyclone 
			laboratories</td>
			<td>SH30265.018</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>&#946;-Glycerophosphate</td>
			<td>Sigma</td>
			<td>G5422</td>
			<td>Osteoblastogenesis</td>
		</tr>
		<tr>
			<td>Caltrate (CAL)</td>
			<td>Wyeth</td>
			<td>L96625</td>
			<td>Animal interventation</td>
		</tr>
		<tr>
			<td>C57BL/6 mice</td>
			<td>SLAC Laboratory 
			Animal Co. Ltd.</td>
			<td>Random</td>
			<td>Ainimal preparation</td>
		</tr>
		<tr>
			<td>Dexamethsome</td>
			<td>Sigma</td>
			<td>D4902</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Sigma</td>
			<td>D2438</td>
			<td>Cell frozen</td>
		</tr>
		<tr>
			<td>Ethylene Diamine Tetraacetic Acid (EDTA)</td>
			<td>Sangon Biotech</td>
			<td>60-00-4</td>
			<td>Samples treatmnet</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>FL-24562</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>Gushukang granules</td>
			<td>kangcheng companyin china</td>
			<td>Z20003255</td>
			<td>Herbal prescription</td>
		</tr>
		<tr>
			<td>Light microscope</td>
			<td>Olympus BX50</td>
			<td>Olympus BX50</td>
			<td>Images for osteoclastogenesis</td>
		</tr>
		<tr>
			<td>L-Ascorbic acid 2-phosphate sequinagneium slat hyclrate</td>
			<td>Sigma</td>
			<td>A8960-5G</td>
			<td>Osteoblastogenesis</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Leica</td>
			<td>DMI300B</td>
			<td>Osteocast and osteoblast imagine</td>
		</tr>
		<tr>
			<td>M-CSF</td>
			<td>Peprotech</td>
			<td>AF-300-25-10</td>
			<td>Osteoclastogenesis</td>
		</tr>
		<tr>
			<td>&#924;icro-CT</td>
			<td>Scanco 
			Medical AG</td>
			<td>&#956;CT80 radiograph microtomograph</td>
			<td>Bone Structural analsysis</td>
		</tr>
		<tr>
			<td>RANKL</td>
			<td>Peprotech</td>
			<td>11682-HNCHF</td>
			<td>Osteoclastogenesis</td>
		</tr>
		<tr>
			<td>Sprague Dawley</td>
			<td>SLAC Laboratory 
			Animal Co. Ltd.</td>
			<td>Random</td>
			<td>Blood serum collection</td>
		</tr>
		<tr>
			<td>Tartrate-Resistant Acid Phosphate (TRAP) Kit</td>
			<td>Sigma-Aldrich</td>
			<td>387A-1KT</td>
			<td>TRAP staining</td>
		</tr>
	</tbody>
</table>

preparation of gushukang (gsk) granules for in vivo and in vitro experiments

中草药在治疗许多疾病(如绝经后骨质疏松症)方面起着替代作用。古树康(GSK)颗粒,在中国销售的处方,在治疗POP具有骨保护作用。在给药到身体之前,通常需要一个标准的制备程序,其目的是促进从生草药中释放活性成分,增强药理作用以及治疗效果。本研究提出了在体内和体外实验测定中使用葛兰素素梅粒的详细方案。作者首先提供了一个详细的协议,以计算动物适合剂量的颗粒进行体内调查:称重,溶解,储存和给药。其次,本文介绍了微CT扫描和骨骼参数测量方案。评估了样品制备、运行微CT机的规程和骨参数的定量。第三,制备含血清的GSK颗粒,提取含药物血清进行体外骨细胞生成和骨细胞生成。葛兰素史克颗粒连续三天每天对大鼠进行两次胃内施用。然后收集血液,离心,灭活,并过滤。最后,对血清进行稀释,用于实施骨细胞生成和骨细胞生成。此处描述的协议可被视为草药处方药(如颗粒)的药理学研究的参考。

微CT扫描结果表明,与盐水对照小鼠相比,OVX小鼠骨质流失显著(图1A)。葛兰素史克颗粒的介入(90天)大大提高了BMD,特别是在GSKM组(图1B)。骨骼结构参数(如 BMD、BV/TV、Tb.N 和Tb.Th)进行了量化。GSK颗粒处理导致BMD、BV/TV、Tb.N和Tb.Th增加,但Tb.Sp(图1C)减少。
与对照小鼠相?...

Watch this Scientific Journal Video about Preparation Of Gushukang GSK Granules for In Vivo and In Vitro Experiments at JoVE.com

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

本文为制备古树康颗粒的工作溶液和用于体外实验的含有血清的GSK颗粒提供了详细的方案。该协议可应用于草药的药理学研究以及体内和体外实验的处方。

古树康 (GSK) 颗粒的制备,用于 Vivo 和体外实验

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

该协议为体内研究提供了颗粒的工作溶液，以及用于体外实验的含颗粒血清的制备。该技术的一个优点是，由于材料成本低，为实验创造的动物成本低，该方法很容易重复。首先向单个50毫升管中加入8、16和24克G古仓（或葛兰水/地王）颗粒。在每个管中加入48毫升的水，并摇动管子以完全溶解颗粒。然后，将1.6片补充维生素D3的碳酸钙放入另一个50毫升的管中，然后用震动将片剂溶解在48毫升的盐水中。对于胃内给给的溶液，首先轻轻而牢固地手动约束一个月大，女性，C57黑6鼠标两到三分钟。当鼠标平静下来后，用0.24毫升的葛口针装上一个0.24毫升的葛子，然后通过嘴的一侧将加夫针放入小鼠，直到感觉到针头的阻力，表明针头已经到达胃部。将整个工作溶液的体积注入动物。然后，收回格瓦奇针，将鼠标返回到其笼子，而其他小鼠正在服用葛兰素史克，然后以相同方式向每只小鼠注射0.24毫升碳酸钙和维生素D3溶液。对于含葛兰素史克的血清采集，在将适当浓度的葛兰素史克（GSK）交付给大鼠后，如刚刚证明的，在上次葛兰素史克颗粒给药后一小时，确认对手趾捏缺乏反应。做一个腹部皮肤和腹腔切口，并暴露腹部到胸部的底部。使用纸巾去除腹部主动脉的结缔组织以清楚地暴露血管，并使用装有 22 量针的 10 毫升注射器从主动脉中抽血。将血液转移到无菌的15毫升管中，并在室温下直立放置管30至60分钟。当血液凝固时，将管子离心分离出血清，并转移多达六只大鼠的所有血清到新的50毫升管中。在56摄氏度的水浴中，在56摄氏度的水浴中分离血清成分之前，摇动管子混合血清，30分钟。然后，通过0.22微米大小的亲水多聚酯硫酮注射器过滤器过滤血清，在零下80摄氏度下储存长达一年。对于体外骨质疏松发生，稀释三个剂量的含葛兰素史克血清在一比四的比例与最小的鹰的介质补充L-谷氨酰胺，核糖核酸，和脱氧核糖核酸。在96井的盘子里，将每根稀释的200微升加入骨髓巨噬细胞培养的每个井中。然后，通过用巨噬细胞聚落刺激因子和受体活化剂刺激每一个井来刺激骨细胞最后生成，以进行核因子-卡帕B配体。对于体外骨细胞生成，在24井板骨细胞细胞培养的每个井中加入两毫升含有含葛兰素史克的稀释血清。微计算断层扫描结果表明，与盐碱对照小鼠相比，奥瓦化小鼠的骨质流失显著。90天的葛兰素史克颗粒治疗干预可大大提高骨髓密度，特别是在中剂量葛兰素史克治疗的动物中。葛兰素史克颗粒治疗还导致骨质增加，骨量超过总体积，和眼骨厚度，但骨骨分离减少。与对照动物相比，耐酸磷酸酶（TRAP）染色表明，与对照动物相比，奥瓦里切除小鼠的骨细胞数量增加，而葛兰素史克颗粒治疗则减少 TRAP阳性骨质分化。与从对照动物收集的血清相比，含葛兰素史克含颗粒血清也减少了 TRAP 阳性骨细胞数。针头成功渗透到腹部主动脉对于保证从每只大鼠中采集足够量的血液至关重要。这种方法的成功执行应分配15至20毫米的血液积累，足够的材料用于体外实验。

Research

JoVE Journal

Medicine

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

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