本研究项目由（1）国家自然科学基金（81973877 82174408）、（2）国家重点研发计划（2020YFE0201600）、（3）上海市重点研究中心建设项目（2022ZZ01009）、（4）上海市医院中药制剂产业转化协同创新中心、（5）上海中医药大学预算内研究项目（2021LK047）资助。

本方案以益奇节度狙为样本，描述了骨肉瘤模型小鼠的详细管饲技术。本实验采用4周龄雄性无胸腺小鼠（BALB/c裸体），饲养在上海中医药大学实验动物中心SPF级育种室。所有动物实验均经上海中医药大学动物管理委员会批准，符合《实验动物伦理规范》（动物伦理批准号:PZSHUTCM2304060007），严格按照国际实验动物相关要求进行。
1. 材料
<ol><li>获取以下材料:中草药（人参 42 g、 Hedyotis diffusa 52.5 g、带刺黄芩草本 31.5 g）、1 mL 注射器和 12G/55 mm 肘部管饲针（图 1）。</li>
</ol>2.剂量测定
<ol><li>按以下方式计算日用量:标准体重60kg的成虫日用量为人参12g、 弥漫蓿 15g、倒刺黄芩草9g，共36g即饮药，即0.6g即食药/kg体重。参考标准体重60kg的成虫与标准体重25g的小鼠11的等效剂量12倍、低剂量6倍、高剂量24倍，以及我们的初步数据，本研究中对小鼠使用剂量比例为20倍的益齐节度汤， 因此，小鼠的剂量为12g即食药物/kg体重。</li>
	<li>使用以下公式计算每只小鼠的剂量: 小鼠剂量（站立体重）=成人剂量（站立体重）×20（指定等效剂量比）。 每只小鼠的日剂量为0.3g[（12g即食药物/ 1000g体重）x 25g体重]在200μL中，终浓度为1.5g/ mL。</li>
	<li>计算强饲给药28天的益琦节度汤组小鼠（n=7）的总煎药为58.8g。将其浓缩至 39.2 mL，以达到 1.5 g/mL 的最终浓度。</li>
	<li>考虑到煎煮和管饲阶段可能发生的药物损失，为了方便计算，制备共126g（人参 42g，Hedyotis diffusa 52.5g，带刺黄芩草31.5g）煎药，浓缩成最终体积为84mL。</li>
</ol>3. 细胞来源和培养
<ol><li>从ATCC细胞库购买人源性143B骨肉瘤细胞。</li>
	<li>在 10 cm 细胞培养皿中将 1 x 106 143B 细胞接种在含有 10% FBS、100 U/mL 青霉素和 100 μg/mL 链霉素的 10 mL MEM 培养基中。</li>
	<li>在37°C下在5%CO2 培养箱中培养143B细胞，直至细胞80%-90%汇合。</li>
</ol>4. 建模方法
<ol><li>收集 80% 汇合培养的 143B 细胞，重悬于 6 mL 含 10% 血清的 MEM 培养基中，并以 1:1 的体积比与基底膜基质凝胶混合，制成终浓度为 1 x 107 个细胞/mL 的细胞悬浮液。</li>
	<li>将 10 μL 细胞悬液注射到每只无胸腺小鼠的左胫骨中，以建立 原位 异种移植骨肉瘤小鼠模型，如我们之前的出版物12 中所述。步骤简述如下。<ol><li>用PBS洗涤细胞2次。用 1.5 mL 0.25% 胰蛋白酶使细胞胰蛋白酶消化 3 分钟。加入 6 mL 含 10% 血清的 MEM 培养基。将细胞收集在 15 mL 离心管中。</li>
		<li>将 20 μL 细胞悬液吸入细胞计数板的腔室中，并使用自动细胞计数器计算细胞浓度。在室温下以800× g 离心细胞5分钟。</li>
		<li>用移液管弃去上清液，将细胞沉淀重悬于8.5mg / mL基底膜基质中，并将其置于冰上。</li>
		<li>通过将小鼠暴露于2%异氟烷和98%氧气（氧流速，2L / min）来麻醉小鼠。在眼睛上涂抹少量兽医软膏，以防止麻醉时干燥。</li>
		<li>在通风良好的地方进行整个过程。在骨肉瘤细胞注射前，通过轻轻触摸脚部，确保每只小鼠处于深度麻醉状态;如果鼠标仍有反应，例如抽搐或抽搐。如果鼠标仍有反应（如抽搐或抽搐），请等待更长的时间，直到这些反应消失。</li>
		<li>将鼠标置于仰卧位。用拇指和食指握住鼠标的脚踝，并用三轮交替的甜菜碱和70%乙醇拭子对胫骨注射部位进行消毒。</li>
		<li>将针头连接到 1 mL 注射器上，并将针尖指向平行于胫骨轴线的注射部位。经皮插入针头，在胫骨远端钻一个孔，穿过胫骨平台。</li>
		<li>将先前放在冰上的骨肉瘤细胞悬液加载到微量注射器中，并用载有骨肉瘤细胞的微量注射器替换胫骨中的 1 mL 注射器。缓慢将约10μL（1×10,7个细胞/ mL）骨肉瘤细胞悬液注射到每个无胸腺小鼠的胫骨中。</li>
		<li>从注射部位取出微量注射器，用棉签按压注射部位约30秒。将鼠标放回干净的笼子中，观察其恢复10分钟。</li>
	</ol></li>
</ol>5.中药管饲配方的制备（图2）
<ol><li>按照步骤2计算剂量。</li>
	<li>将除昂贵的药材人参外的所有药材浸泡在比药材表面高3厘米的饮用水中30分钟，然后如下所述进行2次煎煮。</li>
	<li>通过在强火上加热至沸腾，然后在温和的火上加热15-25分钟来进行第一次煎剂。轻度火灾是指温度通常在 80 °C 至 100 °C 之间的低功率热量，而强火是指通常在 200 °C 至 300 °C 之间的高功率热量。</li>
	<li>将汤液倒入一个容器中。</li>
	<li>通过在饮用水中加热药材进行第二次煎剂，其体积刚好足以淹没药物表面，在强火上煮沸，然后在温和的火上加热15-25分钟。</li>
	<li>将煎剂倒入与步骤5.4相同的容器中。</li>
	<li>将42克人参分别浸泡和煎煮，饮用水比药面高3厘米，因为它很贵。</li>
	<li>通过在强火上加热60分钟来执行浸泡过的人参的第一次煎煮。 将煎剂倒入与步骤5.4相同的容器中。</li>
	<li>在饮用水中进行人参的第二次煎煮，其体积刚好足以淹没药物表面，在温和的火上加热40分钟。将煎剂倒入与步骤5.4相同的容器中。 注意:用于煎剂的容器应具有化学稳定性，例如砂锅菜（临床实践中最常用的）、陶瓷、玻璃和不锈钢，以避免与药物发生化学反应。所有从不同步骤获得的汤液必须放入同一容器中。</li>
	<li>使用用橡皮筋固定在量杯口的三层纱布混合和过滤汤液。</li>
	<li>将过滤后的汤剂在锅中用强火煮沸，然后换成温和的火，保持温和的火，直到达到目标浓度（终体积为84mL，终浓度为1.5g/mL）。</li>
	<li>根据胃内给药所需的剂量制备浓缩汤剂的21mL等分试样1周，并将等分试样储存在-20°C的冰箱中。</li>
</ol>6.使用强饲装置对小鼠进行管饲
<ol><li>将鼠标放在鼠标笼的表面或其他较粗糙的表面上。</li>
	<li>用右手的拇指和食指抓住鼠标的尾巴。然后，用左手的拇指、食指和中指抓住鼠标的脖子。</li>
	<li>用左手的另外两个手指握住鼠标尾巴，使鼠标的头部、颈部和身体成一条直线。 注意: 确保在整个抓取过程中保持鼠标放松。如果鼠标一直挣扎着移动，则应执行抓取过程，直到鼠标放松。</li>
	<li>右手握住管饲装置，为体重为20g的小鼠（100μL汤剂配方/ 10g体重的小鼠）加载200μL汤剂配方。</li>
	<li>将管饲针从角落插入口腔，并将舌头推到小鼠的上颚上。</li>
	<li>将鼠体保持在同一方向，并沿着食道轻轻地将管饲针推向胃部，直到管饲针留在外面约 10 毫米。 注意:当管饲针进入食道时，有一种空心的感觉。插入小鼠的管饲针的长度应测量到胸骨顶部。管饲针的总长度为60mm（图3），因此，在将汤剂注射到小鼠胃中之前，应将约50mm的管饲针插入小鼠体内。</li>
	<li>将汤剂配方注射到小鼠的胃中。遇到阻力或液体从嘴角流出意味着管饲针错位，必须避免。此外，如果汤剂配方较浓，则必须减慢注射速度。</li>
	<li>松开鼠标并观察它们 10 分钟。如果没有观察到呼吸异常，则管饲成功，并将小鼠放回笼中（图4）。</li>
</ol>7.疗程
<ol><li>为探究益奇节度汤的治疗效果，设置对照组和益奇节度度汤组。对于对照组中的小鼠，给予生理盐水，每天一次，持续28天。对于益奇洁度汤组的小鼠，每天施用益汤28天。</li>
</ol>8.不良反应
<ol><li>仔细观察小鼠被放回笼子后的状态，包括体重减轻和由于食物无法进入胃部而导致的精神状态下降。</li>
	<li>由于食物引起的气管阻塞，小鼠会出现呼吸困难和呼吸急促，并会咳嗽以试图清除气管。如果在管饲过程中观察到任何这些症状，请立即停止管饲并对小鼠实施安乐死。</li>
</ol>

骨肉瘤小鼠的草药煎剂制剂和灌胃标准化

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作者声明他们没有什么可披露的。

处方药材在中药中的临床疗效很大程度上与给药剂量和剂型有关;因此，在临床实践中，建立标准化的煎剂策略和熟练的给药过程以及提高实验结果的可靠性是必要的。
本研究首先为中药临床实践中药材标准化汤剂提供了详细的策略，便于引导人们科学用药，也切实提高了中药的汤剂质量和临床疗效;因此，一旦实现标准化，将提高草药疗效在临床实践中的准确性和一致性。
管饲策略在临床前研究中作为口服摄入方法被广泛应用，适用于各种实验动物，包括小鼠、大鼠和兔子，具有易于给药、准确剂量控制、能够模仿临床给药途径等优点13。然而，如果动物在没有麻醉的情况下被固定在不正确的位置，它们会挣扎，这可能会威胁研究人员的健康并降低实验的可靠性。因此，建立标准化的管饲方法对于减少这些可能性，避免汤剂误入食道或造成食道损伤，进而导致动物死亡和实验失败具有重要意义。
在这里，我们选择了管饲途径将汤剂配方输送到骨肉瘤模型小鼠中，因为它使我们能够精确测量剂量并保证在整个实验过程中一致的药物输送。为了制备煎剂配方，我们首先根据体重为60kg的成人用量的20倍的比例计算小鼠的即饮药量;浸泡，加热至沸腾煎煮2次;过滤浓缩，并将煎剂等分储存在-20°C下。 为了进行管饲给药，将200μL浓缩汤剂式装入管饲装置中，将管饲针插入口中，轻轻地将管饲针推向胃，然后将汤剂注射到小鼠的胃中。我们的发现之一是在接受通过强饲法接受益祁节度汤剂汤剂的组中观察到肿瘤生长的显着抑制。与对照组相比，益琦节度汤组的肿瘤体积明显减少。这些结果表明，管饲策略有效地将汤剂配方的活性成分输送到靶位点，从而产生有效的抗肿瘤作用。此外，我们通过在整个实验期间监测小鼠的一般状况和体重来评估管饲策略的安全性。益奇洁度汤管饲组与对照组体重变化或毒性征象差异无统计学意义，说明汤剂管饲给药耐受性良好，不引起明显不良反应。
尽管管饲策略被证明可以有效地将汤剂配方输送到实验动物，但重要的是要认识到与这种给药方法相关的潜在不良反应和某些局限性。为避免食物引起的气管阻塞，请在灌胃期间和之后密切观察小鼠状态，例如呼吸困难、呼吸急促和咳嗽。一旦出现其中任何一个，立即停止管饲并在水平硬桌面上进行胸外按压;此外，任何严重情况都需要立即获得兽医帮助。此外，我们需要密切关注以下限制。首先，强饲法可能会在动物中引起压力，从而可能影响它们的整体生理机能和对治疗的反应。然而，我们采取了一些措施，通过使用小的喂食针并在管饲过程中轻柔地处理小鼠来尽量减少压力。此外，管饲法只能由经过专门培训和合格的人员进行，以确保使用标准化技术来避免对动物可能造成的伤害14.其次，汤剂配方中的活性成分发挥抗肿瘤作用的确切机制尚不清楚。管饲策略可能会改变草药化合物的药代动力学和药效学，从而可能影响其生物利用度和治疗效果。未来的研究应侧重于阐明通过管饲法递送的汤剂配方的抗肿瘤作用的确切机制。此外，研究报告称，在长期管饲过程中应用异氟醚麻醉可减少汤剂的不完全摄入和由于不熟练的管饲技术而导致的动物死亡15。同时，在汤剂中加入食用油可减少与管饲相关的动物应激16。
综上所述，我们提供了中草药的标准化煎煮工艺和熟练的小鼠灌胃策略。这些努力不仅避免了实验小鼠可能的食道损伤和死亡，而且避免了药物溢出，确保每只实验动物给予等量的草药，从而提高了实验结果的准确性和一致性，也提高了治疗效果。本研究证明了管饲策略在骨肉瘤小鼠模型中施用中药汤剂的有效性和安全性。这些结果凸显了这种给药方法在治疗骨肉瘤中的潜力，并为进一步研究优化中药管饲策略奠定了基础。此外，未来的研究应调查汤剂配方中活性成分的确切作用机制，并探索与其他治疗方式的潜在协同作用，以提高骨肉瘤患者的治疗效果。因此，这些发现揭示了这种给药方法的潜在益处和局限性，并为其在骨肉瘤治疗中的应用提供了宝贵的见解。

汤剂是中药和药液中最常用的剂型1,2。中药的汤剂配方是将草药浸泡后在水中煎煮，然后过滤以丢弃渣滓。
由于汤剂配方可以根据临床需要进行个体化，特别是符合中医循证治疗的特点，这种经口给药的剂型在临床实践中已有数千年的应用，具有特定且不可替代的优势3,4.汤剂配方的好坏与临床疗效直接相关。因此，必须根据标准化的汤剂方法原理制备汤剂，以确保有效成分得到最大程度的提取和保护3。同时，即饮用药是临床实践中使用的生中草药的主要形式;处方即饮药的质量标准和生产方法必须符合《中国药典》第二版的规定。此外，处方的即食药必须在冷饮水中浸泡至少30分钟，然后才能在特殊的药锅中加热5。研究表明，大部分有效成分可通过煎煮两次来提取，《医疗机构中药煎剂室管理操作规范》也规定煎煮两次4,6,7。如果开了特殊的中草药，例如质地坚硬或滋补品的中草药，建议延长煎煮时间。与汤剂直接接触的汤器皿和储存容器，从浸泡处方即食药物的容器开始，应具有化学稳定性并有盖，例如砂锅菜、陶瓷、玻璃和不锈钢。但是，不应使用铝和普通塑料制品，而应禁止使用铁和其他腐蚀性器皿，以避免可能的化学反应，这会降低治疗效果，甚至产生有害影响4,6,7。
由于进行临床实验的局限性，携带人类疾病的小动物，如小鼠，经常被用于医学研究，以探索不同干预措施的治疗效果和综合机制，包括中药的汤剂配方8,9;然而，与人类不同的是，汤剂配方应每天配制，混合在一起，并在临床实践中每天新鲜给药2次。动物无法自愿按时按规定剂量服药，目前与中药相关的动物实验大多是以管饲法口服的汤剂给药。用于体内动物实验的汤剂配方应提前制备，等分，在-20°C下储存，并在使用前解冻3,4,10。此外，不合格的动物固定和管饲针操作会增加不必要的伤害和负面情绪的风险。因此，作为经口给药的重要方法，熟练的管饲技术对于避免可能的食管损伤和药物溢出尤为重要，从而保证每只模式动物接受等量的处方草药，以达到准确的实验结果。在这里，我们为中药的汤剂配方提供了一个灌胃方案。

<table><tbody><tr><td>143B cell line</td><td>ATCC</td><td>CRL-8303</td><td>osteosarcoma cell line</td></tr><tr><td>Anesthesia machine</td><td>Shenzhen RWD Life Technology Co.,Ltd</td><td>R500IP</td><td>The Equipment of Anesthesia mice</td></tr><tr><td>Automatic cell counter</td><td>Shanghai Simo Biological Technology Co., Ltd</td><td>IC1000</td><td>Counting cells</td></tr><tr><td>BALB/c athymic mice</td><td>Shanghai SLAC Laboratory Animal Co, Ltd.</td><td>Male</td><td>Animal</td></tr><tr><td>Barbed skullcap herb</td><td>Shanghai Yanghetang Traditional Chinese Medicine Tablet Co., Ltd.</td><td>20230606</td><td>Decoction-ready medicines&amp;nbsp;</td></tr><tr><td>Basement Membrane Matrix</td><td>Shanghai Uning BioscienceTechnology Co., Ltd</td><td>356234, BD, Matrigel</td><td>re-suspende cells</td></tr><tr><td>Centrifuge tube (15 mL)</td><td>Shanghai YueNian Biotechnology Co., Ltd</td><td>430790, Corning</td><td>Centrifuge the cells</td></tr><tr><td>Elbow gavage needle (12-gauge/ 55 mm)</td><td>RWD Life Science Co., Ltd.</td><td>C21014-12</td><td>Component of gastric perfusion device</td></tr><tr><td>Gauze</td><td>Haishi Hainuo Group Co., Ltd.</td><td>LC45</td><td>Decoction filter</td></tr><tr><td>Ginseng</td><td>Shanghai Wanshicheng Pharmaceutical Co., Ltd.</td><td>20220704-2</td><td>Decoction-ready medicines&amp;nbsp;</td></tr><tr><td>Hedyotis diffusa</td><td>Shanghai Yanghetang Traditional Chinese Medicine Tablet Co., Ltd.</td><td>2023071107</td><td>Decoction-ready medicines&amp;nbsp;</td></tr><tr><td>isoflurane</td><td>Shenzhen RWD Life Technology Co., Ltd</td><td>VETEASY</td><td>Anesthesia mice</td></tr><tr><td>Micro-volume syringe</td><td>Shanghai high pigeon industry and trade Co., Ltd</td><td>0-50 &amp;mu;L</td><td>Inject precise cells into the tibia</td></tr><tr><td>Phosphate-buffered saline</td><td>Beyotime Biotechnology</td><td>ST447</td><td>wash the human osteosarcoma cells</td></tr><tr><td>Rubber bands</td><td>Shanghai Hengfei Biotechnology Co., Ltd.</td><td>XPJ</td><td>Fixing gauze on the measuring cup</td></tr><tr><td>Syringe (1 mL )</td><td>Shanghai Mishawa Medical Industry Co., Ltd.</td><td>SBM0040</td><td>Component of gastric perfusion device</td></tr><tr><td>Trypsin (0.25%)</td><td>Shanghai YueNian Biotechnology Co., Ltd</td><td>25200056, Gibco</td><td>trypsin treatment of cells</td></tr></tbody></table>

gavage strategy for decoction formula of traditional chinese medicine in osteosarcoma model mice

汤剂是中医中最常用的剂型，经口给药在临床实践中应用了数千年，其特点是见效快、易吸收、根据患者具体症候进行个体化治疗。汤剂配方的好坏直接关系到中药的临床疗效;因此，汤剂配方的标准化过程对于避免主观因素造成的汤剂质量差异具有重要意义。同时，由于进行临床实验的局限性，携带人类疾病的小动物，如小鼠，经常被用于医学研究，以探索不同干预措施的治疗效果和综合机制，包括中药的汤剂配方。因此，作为一种重要的经口给药方法，熟练的管饲技术对于避免潜在的食管损伤和药物溢出尤为重要，这将确保对每只模型动物施用等量的药物，从而获得准确的实验结果。此外，标准化的汤剂配方制备方法和熟练的管饲策略对于保护动物福利和尽量减少使用动物数量是必要的。在此，我们以益奇节度汤剂在骨肉瘤小鼠模型中报告了煎剂配方和管饲技术的详细标准化过程。通过肿瘤体积评估疗效。该方案将最大限度地保护动物，提高研究数据的可靠性，从而为未来研究中药 汤剂体内的治疗效果和分子机制提供有效的策略。

以胫骨内异种移植骨肉瘤小鼠模型为研究对象，将143B骨肉瘤细胞注射到无胸腺小鼠胫骨中，测定了益奇节度汤对骨肉瘤 体内 生长的抑制作用。从143B注射的第二天开始，以强饲法给益祺节度汤组小鼠施用益祁节度汤28 d。对照组小鼠给予生理盐水。所有小鼠在28天后被安乐死。结果显示，与对照组相比，益祁节度汤组的肿瘤体积明显减少（图5）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66177/66177fig01.jpg"> 图 1:煎剂耗材。 即饮药包括（A）人参，（B）Hedyotis diffusa，（C）带刺黄芩草，（D）刻度量杯，（E）橡皮筋，（F）纱布，（G）用橡皮筋在量杯口固定三层纱布。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66177/66177fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66177/66177fig02.jpg"> 图2:中药汤剂配方的制备。 （A） 除人参外的可煎药浸泡 30 分钟。 （B） 人参单独浸泡 30 分钟。 （C） 人参单独煎煮。（四）中药过滤汤剂配方。（五）中药浓缩汤剂的贮存。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66177/66177fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66177/66177fig03.jpg"> 图 3:灌胃耗材。 常规的 1 mL 注射器（左图）和 1 mL 注射器（原装针头）替换为 12/55 mm 肘部胃灌注针以设置管饲装置（右图）。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66177/66177fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66177/66177fig04.jpg"> 图4:小鼠管饲给药程序。 （A） 鼠标在笼子表面。（B） 用一只手抓住老鼠尾巴。（C-D）用一只手固定鼠标。（E） 从鼠标嘴插入针头。（F） 管饲法管理结束。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66177/66177fig04large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66177/66177fig05.jpg"> 图5:汤剂对骨肉瘤小鼠模型的抑制作用。 （A）从对照组和益奇节度汤干预组收集的骨肉瘤样本。误差线代表标准差 （n=7） （B） 对照组 （Ctrl） 组和益琦节度汤干预组之间肿瘤体积的未配对 t 检验。益祁节度汤干预28天可显著抑制肿瘤体积（p<0.05）。误差线代表标准差 （n=7）。（C）整个实验期间益奇节度汤管饲组和对照组小鼠的体重。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66177/66177fig05large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>

作者聚焦:用于增强动物研究的标准化草药汤剂方案

该方案以骨肉瘤小鼠模型中的益奇节度汤提供了详细的汤剂配方和管饲技术标准化流程。它描述了动物保护，提高了研究数据的可靠性，为研究汤剂 在体内的治疗效果和分子机制提供了有效的策略。

中药中药汤剂在骨肉瘤模型小鼠中的强饲策略

Decoction formula is the most commonly used dosage form in traditional Chinese medicine and applied in clinical practice for thousands of years by ...

本研究为循序渐进的预防准备提供了详细和标准化的方案，为有需要的人提供了参考。我们的研究确保每只动物的剂量相等，以产生准确的结果。我们的研究旨在最大限度地保护动物，并提高汤剂制剂研究结果的可靠性。

gavage-strategy-for-decoction-formula-traditional-chinese-medicine

Universidad San Sebastian

Research

JoVE Journal

Medicine

Gavage Strategy for Decoction Formula of Traditional Chinese Medicine in Osteosarcoma Model Mice

557 次观看.  Shanghai University of Traditional Chinese Medicine. 本研究为循序渐进的预防准备提供了详细和标准化的方案，为有需要的人提供了参考。我们的研究确保每只动物的剂量相等，以产生准确的结果。我们的研究旨在最大限度地保护动物，并提高汤剂制剂研究结果的可靠性。

视频: 作者聚焦:用于增强动物研究的标准化草药汤剂方案

Practical Considerations in Studying Metastatic Lung Colonization in Osteosarcoma Using the Pulmonary Metastasis Assay

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung colonization in osteosarcoma (OS) by fluorescence microscopy. This article provides a detailed description of the protocol, and discusses examples of obtaining image data on metastatic growth using widefield or confocal fluorescence microscopy platforms. The flexibility of the PuMA model permits researchers to study not only the growth of OS cells in the lung microenvironment, but also to assess the effects of anti-metastatic therapeutics over time. Confocal microscopy allows for unprecedented, high-resolution imaging of OS cell interactions with the lung parenchyma. Moreover, when the PuMA model is combined with fluorescent dyes or fluorescent protein genetic reporters, researchers can study the lung microenvironment, cellular and subcellular structures, gene function, and promoter activity in metastatic OS cells. The PuMA model provides a new tool for osteosarcoma researchers to discover new metastasis biology and assess the activity of novel anti-metastatic, targeted therapies.

The goal of this article is to provide a detailed description of the protocol for the pulmonary metastasis assay (PuMA). This model permits researchers to study metastatic osteosarcoma (OS) cell growth in lung tissue using a widefield fluorescence or confocal laser-scanning microscope.

应用肺转移法研究骨肉瘤转移性肺定植的实用思考

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung ...

科研

癌症研究

A Preclinical Mouse Model of Osteosarcoma to Define the Extracellular Vesicle-mediated Communication Between Tumor and Mesenchymal Stem Cells

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under the influence of specific environmental signals, these adult stem cells can release paracrine mediators leading to accelerated tumor growth and metastasis. Defining the crosstalk between tumor and MSCs is of primary importance to understand the mechanisms underlying cancer progression and identify novel targets for therapeutic intervention. 
Cancer cells produce high amounts of extracellular vesicles (EVs), which can profoundly affect the behavior of target cells in the tumor microenvironment or at distant sites. Tumor EVs enclose functional biomolecules, including inflammatory RNAs and (onco)proteins, that can educate stromal cells to enhance the metastatic behavior of cancer cells or to participate in the pre-metastatic niche formation. In this article, we describe the development of a preclinical cancer mouse model that enables specific evaluation of the EV-mediated crosstalk between tumor and mesenchymal stem cells. First, we describe the purification and characterization of tumor-secreted EVs and the assessment of the EV internalization by MSCs. We then make use of a multiplex bead-based immunoassay to evaluate the alteration of the MSC cytokine expression profile induced by cancer EVs. Finally, we illustrate the generation of a bioluminescent orthotopic xenograft mouse model of osteosarcoma that recapitulates the tumor-MSC interaction, and show the contribution of EV-educated MSCs to tumor growth and metastasis formation. 
Our model provides the opportunity to define how cancer EVs shape a tumor-supporting environment, and to evaluate whether blockade of the EV-mediated communication between tumor and MSCs prevents cancer progression.

Direct injection of cancer-derived extracellular vesicles (EVs) leads to reprogramming of bone marrow supporting tumor progression; however, which cells mediate this effect is unclear. Herein, we describe a step-by-step protocol to investigate EV-mediated tumor-mesenchymal stem cell (MSC) interactions in vivo, revealing a crucial role for EV-educated MSCs in metastasis.

骨肉瘤前小鼠模型对肿瘤与间充质干细胞细胞外囊泡介导通信的定义

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under ...

Modeling Osteosarcoma Using Li-Fraumeni Syndrome Patient-derived Induced Pluripotent Stem Cells

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including osteosarcoma--one of the most frequent primary non-hematologic malignancies in the childhood and adolescence. Therefore, LFS provides an ideal model to study this malignancy. Taking advantage of iPSC methodologies, LFS-associated osteosarcoma can be successfully modeled by differentiating LFS patient iPSCs to mesenchymal stem cells (MSCs), and then to osteoblasts--the cells of origin for osteosarcomas. These LFS osteoblasts recapitulate oncogenic properties of osteosarcoma, providing an attractive model system for delineating the pathogenesis of osteosarcoma. This manuscript demonstrates a protocol for the generation of iPSCs from LFS patient fibroblasts, differentiation of iPSCs to MSCs, differentiation of MSCs to osteoblasts, and in vivo tumorigenesis using LFS osteoblasts. This iPSC disease model can be extended to identify potential biomarkers or therapeutic targets for LFS-associated osteosarcoma.

Here, we present a protocol for the generation of induced pluripotent Stem Cells (iPSCs) from Li-Fraumeni Syndrome (LFS) patient derived fibroblasts, differentiation of iPSCs via mesenchymal stem cells (MSCs) to osteoblasts, and modeling in vivo tumorigenesis using LFS patient-derived osteoblasts.

应用 Fraumeni 综合征诱导多潜能干细胞建模骨肉瘤

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including ...

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with diagnostic or prognostic difficulties. OS comprises genotypically and phenotypically heterogeneous cancer cells. Bone microenvironment elements are proved to account for tumor heterogeneity and disease progression. Bone extracellular matrix (BEM) retains the microstructural matrices and biochemical components of native extracellular matrix. This tissue-specific niche provides a favorable and long-term scaffold for OS cell seeding and proliferation. This article provides a protocol for the preparation of BEM model and its further experimental application. OS cells can grow and differentiate into multiple phenotypes consistent with the histopathological complexity of OS clinical specimens. The model also allows visualization of diverse morphologies and their association with genetic alterations and underlying regulatory mechanisms. As homologous to human OS, this BEM-OS model can be developed and applied to the pathology and clinical research of OS.

The bone extracellular matrix (BEM) model for osteosarcoma (OS) is well established and shown here. It can be used as a suitable scaffold for mimicking primary tumor growth in vitro and providing an ideal model for studying the histologic and cytogenic heterogeneity of OS.

骨肉瘤的三维骨细胞外基质模型

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

A Syngeneic Orthotopic Osteosarcoma Sprague Dawley Rat Model with Amputation to Control Metastasis Rate

The most recent advance in the treatment of osteosarcoma (OS) occurred in the 1980s when multi-agent chemotherapy was shown to improve overall survival compared to surgery alone. To address this problem, the aim of the study is to refine a lesser-known model of OS in rats with a comprehensive histologic, imaging, biologic, implantation, and amputation surgical approach that prolongs survival. We used an immunocompetent, outbred Sprague-Dawley (SD), syngeneic rat model with implanted UMR106 OS cell line (originating from a SD rat) with orthotopic tibial tumor implants into 3-week-old male and female rats to model pediatric OS. We found that rats develop reproducible primary and metastatic pulmonary tumors, and that limb amputations at 3 weeks post implantation significantly reduce the incidence of pulmonary metastasis and prevent unexpected deaths. Histologically, the primary and metastatic OSs in rats were very similar to human OS. Using immunohistochemistry methods, the study shows that rat OS are infiltrated with macrophages and T cells. A protein expression survey of OS cells reveals that these tumors express ErbB family kinases. Since these kinases are also highly expressed in most human OSs, this rat model could be used to test ErbB pathway inhibitors for therapy.

Here, a syngeneic orthotopic implantation followed by an amputation procedure of the osteosarcoma with spontaneous pulmonary metastasis that can be used for preclinical investigation of metastasis biology and development of novel therapeutics is described.

同源原位骨肉瘤Sprague Dawley大鼠模型，截肢控制转移率

The most recent advance in the treatment of osteosarcoma (OS) occurred in the 1980s when multi-agent chemotherapy was shown to improve overall survival ...

Intratibial Osteosarcoma Cell Injection to Generate Orthotopic Osteosarcoma and Lung Metastasis Mouse Models

Osteosarcoma is the most common primary bone cancer in children and adolescents, with lungs as the most common metastatic site. The five-year survival rate of osteosarcoma patients with pulmonary metastasis is less than 30%.Therefore, the utilization of mouse models mimicking the osteosarcoma development in humans is of great significance for understanding the fundamental mechanism of osteosarcoma carcinogenesis and pulmonary metastasis to develop novel therapeutics. Here, detailed procedures are reported to generate the primary osteosarcoma and pulmonary metastasis mouse models via&#160;intratibia injection of osteosarcoma cells. Combined with the bioluminescence or X-ray live imaging system, these living mouse models are utilized to monitor and quantify osteosarcoma growth and metastasis. To establish this model, a basement membrane matrix containing osteosarcoma cells was loaded in a micro-volume syringe and injected into one tibia of each athymic mouse after being anesthetized. The mice were sacrificed when the primary osteosarcoma reached the size limitation in the IACUC-approved protocol. The legs bearing osteosarcoma and the lungs with metastasis lesions were separated. These models are characterized by a short incubation period, rapid growth, severe lesions, and sensitivity in monitoring the development of primary and pulmonary metastatic lesions. Therefore, these are ideal models for exploring the functions and mechanisms of specific factors in osteosarcoma carcinogenesis and pulmonary metastasis, the tumor microenvironment, and evaluating the therapeutic efficacy in vivo.

The present protocol describes intratibia osteosarcoma cell injection to generate mouse models bearing orthotopic osteosarcoma and pulmonary metastasis lesions.

胫骨肉瘤细胞注射生成原位骨肉瘤和肺转移小鼠模型

Osteosarcoma is the most common primary bone cancer in children and adolescents, with lungs as the most common metastatic site. The five-year survival ...

<meta charset="utf8"></head><body>骨肉瘤 PDX 小鼠模型的方案：研究生长和耐药性

Establishment of Patient-Derived Xenograft Mouse Model with Human Osteosarcoma Tissues

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. Despite the development of new treatment plans in recent years, the prognosis for osteosarcoma patients has not significantly improved. Therefore, it is crucial to establish a robust preclinical model with high fidelity. The patient-derived xenograft (PDX) model faithfully preserves the genetic, epigenetic, and heterogeneous characteristics of human malignancies for each patient. Consequently, PDX models are considered authentic in vivo models for studying various cancers in transformation studies. This article presents a comprehensive protocol for creating and maintaining a PDX mouse model that accurately mirrors the morphological features of human osteosarcoma. This involves the immediate transplantation of freshly resected human osteosarcoma tissue into immunocompromised mice, followed by successive passaging. The described model serves as a platform for studying the growth, drug resistance, relapse, and metastasis of osteosarcoma. Additionally, it aids in screening the target therapeutics and establishing personalized treatment schemes.

<meta charset="utf8"></head><body>作者聚焦：在免疫缺陷小鼠中复制人骨肉瘤进展用于癌症研究

The present protocol describes the method for establishing a patient-derived xenograft (PDX) mouse model using human osteosarcoma tissue.

人骨肉瘤组织患者来源的异种移植小鼠模型的建立

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. Despite the development of new treatment plans in recent years, ...

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.

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 ...

中药中药汤剂在骨肉瘤模型小鼠中的强饲策略

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同源原位骨肉瘤Sprague Dawley大鼠模型，截肢控制转移率

胫骨肉瘤细胞注射生成原位骨肉瘤和肺转移小鼠模型

人骨肉瘤组织患者来源的异种移植小鼠模型的建立

人骨肉瘤组织患者来源的异种移植小鼠模型的建立

人骨肉瘤组织患者来源的异种移植小鼠模型的建立

古树康 (GSK) 颗粒的制备,用于 Vivo 和体外实验