这项研究得到了 NIH/R21CA195002-01A1, 康奈尔妇女协会会长和美国纽约州立大学研究生多样性奖学金的支持。Dr. 卡明斯实验室也在项目期间从康奈尔比较癌症生物训练计划和礼礼和公司获得了资金。

所有的实验方案都得到了康奈尔大学机构动物护理和使用委员会的批准。
1. 手术前准备
注意: 研究小鼠通常是 C57BL 饮食诱发的肥胖背景, 使研究翻译与人类肥胖和胰岛素抵抗有关。在随后的步骤中, 可以对雄性和雌性小鼠进行研究。
<ol>
	<li>在2月的年龄, 将老鼠放在45% 或60% 高脂肪的饮食中2月 (参见材料表)。 注: 老鼠被喂食高脂肪饮食, 以创造一个肥胖和胰岛素抵抗的表型。更短或更长时期的高脂饮食喂养, 可根据该研究的目标使用。</li>
	<li>继续喂养小鼠ad 随意直到手术。如果需要基线测量, 经过7周的高脂饮食喂养, 快速小鼠6小时或一夜, 并采取基线血液样本测量的利益代谢物 (例如,葡萄糖)。</li>
	<li>继续吃高脂肪的小鼠, 允许小鼠在进行假手术或 VSG 前一周的血液回收。在手术前4天将老鼠放在液体饮食上 (见材料表)。 注意: 小鼠在手术前后保持液体饮食, 以清除可能损害愈合反应的微粒物质的消化道。</li>
	<li>在手术前对下列物品进行消毒: 箔、纱布、棉签 (cta) 和手术器械 (止血、操作剪刀、虹膜剪刀、cep、锯齿褐色-Adson cep、胃针、针驱动、弹簧剪刀).</li>
</ol>
2. 垂直袖胃切除术和假手术
<ol>
	<li>在手术当天, 称小鼠为基线体重测量。</li>
	<li>为手术室配备麻醉系统、温水浴和加热垫。将水浴设置为37° c, 用它加热0.9% 盐水溶液进行灌溉。用70% 乙醇清洁手术场, 使用无菌技术打开蒸压的手术包和工具。 注意: 在手术和麻醉恢复过程中, 加热垫用于保持动物的体温。</li>
	<li>使用感应室, 麻醉5% 异氟醚和 O2的流量为1升/分, 确认鼠标在适当的麻醉平面上执行脚趾捏。一旦达到了一致的麻醉平面, 保持1-3% 异氟醚和 O2流量0.6 升/分钟的鼠标。</li>
	<li>一旦鼠标被麻醉, 眼睛药膏 (见材料表) 在其眼中, 以保持他们在手术中潮湿。在这个时候也管理镇痛剂, 例如昔 (2 毫克/千克皮下)。修剪头发从肚脐到腋保持皮肤轻轻地提供紧张。
	<ol>
		<li>由于鼠标的皮肤是薄而细腻的, 注意不要引起皮肤损伤时修剪头发。用聚维酮碘和酒精清洁皮肤。</li>
		<li>不育手套和准备无菌的外科领域和仪器。使用无菌技术进行其余的过程。在老鼠的两侧放置不育的手术窗帘, 准备无菌的外科手术场。使用蒸压锡箔为鼠标创建一个外科悬垂。</li>
		<li>在锡纸上切一个小洞, 允许进入老鼠的腹部。</li>
	</ol>
	</li>
	<li>用虹膜剪刀将皮肤从 mid-abdomen (脐) 切开到剑软骨的水平。识别式阿尔巴, 并使用虹膜剪刀削减沿式阿尔巴的身体壁。用 cta 轻轻地将胃从腹部抬起, 然后直接解剖大网膜, 使其更大的曲率。</li>
	<li>结扎用7-0 单丝可吸收缝合线放置两个连字, 在胃底和脾之间运行的短胃动脉。用弹簧剪刀剪下两个连字。一旦动脉断, 完全取出胃从腹腔。将纱布置于胃下, 用盐水浸湿, 保持组织湿润。</li>
	<li>假程序
	<ol>
		<li>对于假手术, 采用6-0 单丝可吸收缝线和锥形针, 放置一个松散的简单连续缝合方式。从食道右侧 (外科医生右侧) 开始2毫米。将缝合线沿腹侧胃壁放置, 然后沿胃背壁继续。 注: 对于所有的胃操作, 使用单丝可吸收缝合, 以减少感染的风险。此外, 使用针针缝合所有的胃操作, 因为使用切割针头增加缝合的风险 "拉过" 导致胃组织损伤和伤口裂开。</li>
		<li>将针完全通过一个胃壁, 同时放置缝合。确保缝线是平的, 但不能限制胃。用3-4 投掷轻轻地扎下缝合线, 跳到步骤2.9。</li>
		<li>对于 VSG 过程, 请跳过步骤2.7 并继续步骤2.8。</li>
	</ol>
	</li>
	<li>VSG 做法
	<ol>
		<li>对于 VSG 程序, 结扎7-0 单丝可吸收缝线与锥针使用3投掷每个结的胃动脉和静脉突出分支。将连字放在下面 (即, 对胃的较小的曲率) 所预期的横断线。 注意: 预期的横断线起始于 ~ 2 毫米以上 (意思是朝向更大的胃的曲率) 胃的心脏切口和至少2毫米在玛歌 pilcatus 之下并且延伸到胰腺的右叶的近端。通常, 每胃壁有4血管需要结扎;然而, 这可能是不同的小鼠。</li>
		<li>为防止胃切除术中的胃液溢出, 将一条简单的连续缝合线通过6-0 单丝可吸收缝线与锥形针一起穿过两个胃壁。开始缝合线, 以外科医生的右食管和在玛歌 pilcatus 和结束略高于胰腺。</li>
		<li>在缝合线上方放置细尖的止血, 用弹簧剪刀在缝合线和止血之间切割。从不育的外科领域取出断的胃组织。</li></ol></li></ol>使用 cta 清洁血液和糜的胃。
		<li>用一个简单的间断模式, 用6-0 单丝可吸收缝线和锥形针加强胃壁的并列。 注意: 建议采用一种简单的间断缝合模式, 因为它比简单的连续缝合模式提供更安全的闭合。</li>
		<li>在整个手术过程中用盐水冲洗残胃, 以保持组织的湿润和清洁。使用一个20G 胃针连接到一个20毫升的注射器执行洗胃的盐水。使用这种大小, 以确保有足够的压力, 而不会危及胃组织损伤过度的压力或意外的胃组织损害使用常规尖针。</li>
		<li>使用最少20节, 以安全地关闭胃;注意沿食管一侧闭合, 因为这一侧更难进入, 因此往往是一个站点的开裂。</li>
		<li>用 cta 轻轻按压胃部, 确保无渗漏。如果发现泄漏, 在泄漏区域放置额外的简单不连续节点, 然后再次进行泄漏测试。泄漏测试, 直到未检测到泄漏。</li>
		<li>用至少60毫升的盐水做最后彻底的胃灌洗, 以确保没有任何传染性的颗粒物被留下。</li>
	
	
	<li>用 CTA 将胃放回肝脏下的腹腔。将 cta 放在腹腔的背侧, 吸收所有多余的液体。使用钝的18G 针, 注入林格林格的解决方案 (0.5 毫升/-20 毫克/千克恩诺) 直接进入腹腔, 只是在关闭之前。 注意: 这取代了在手术过程中经历的体液损失, 并提供了一种直接应用抗生素到外科手术部位以帮助恢复的方法。</li>
	<li>用6-0 单丝可吸收缝线与锥形针在一个简单的间断模式下闭合腹肌层。然后用6-0 单丝可吸收缝合线在一个简单的连续模式下闭合皮肤层。</li>
	<li>将组织粘合剂贴在皮肤上, 将皮肤折叠在缝合线上, 以掩埋缝线, 使小鼠不能扰乱伤口闭合术。</li>
	<li>关闭异氟醚, 让鼠标恢复在加热垫 10-15 minbefore 返回它的家庭笼。不要让动物无人看管, 直到它恢复知觉并能够移动。</li>
	<li>将动物单独安置, 直到从手术中完全恢复。 注意: 如果在整个研究过程中都能获得准确的食物摄入量, 老鼠应该继续在整个研究中单独居住。有外国身体阻塞的危险, 从吃家庭笼床上用品。动物应该被安置在笼子里没有寝具, 除了嵌套材料提供丰富 (参见材料表)。</li>

3. 操作后鼠标护理和测量
<ol>
	<li>将鼠标返回其主笼, 并将其放在加热垫上。在手术后至少7天内维持小鼠的液体饮食。在手术后2天内施行镇痛药, 如昔 (2 毫克/千克皮下)。 注: 抗生素, 如恩诺 (20 毫克/千克), 可在手术后7天内给予。抗生素被用来减少手术过程中感染的风险, 并且可能或可能不需要, 这取决于整个过程中保持的不育程度。然而, 如果使用抗生素, 必须在同一项研究中对所有小鼠进行管理, 以保持一致性。</li>
	<li>在术后期间评估体重, 食物摄入量, 排便, 活动水平和处置至少每天, 以确保适当的愈合和恢复。术后14天进行术后监测。 注: 手术失败通常在术后或术三天内立即出现。在手术后第二天不进食或饮酒的动物必须严密监测手术并发症。</li>
	<li>术后成功完成后, 定期测量体重和食物摄入量, 确保适当的表型保持正常。</li>
	<li>进行口服葡萄糖耐受试验 (OGTT), 以评估葡萄糖耐受, 葡萄糖刺激胰岛素分泌, 和肠道激素分泌。
	<ol>
		<li>执行 OGTT 在小鼠禁食后6小时, 然后胃小鼠50% 葡萄糖溶液在1克/千克剂量。</li>
		<li>收集尾血样本在 0, 2.5, 5, 15, 30, 60, 120 分钟后, 胃。在这些时间点测量血糖与血糖。在每个时间点收集血清样本, 以测量各种激素, 如胰岛素和 GLP-1。</li>
		<li>安乐死前6小时的快速小鼠。做最后的血样和葡萄糖测量。将最后的葡萄糖测量与基线测量进行比较。安乐小鼠腹腔注射戊巴比妥200毫克/千克剂量。</li>
		<li>使用基线和最终血清样本来测量额外的分析, 并比较术前和手术前后的价值。</li>
	</ol>
	</li>
</ol>

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作者声明没有利益冲突, 除非 Dr. 卡明斯收到了 Eli 和公司的资助。

减肥手术是最有效的长期治疗肥胖和结果的其他健康的好处, 如高率2型糖尿病和高血压缓解1,9,15。小鼠减肥手术模型提供了一个强有力的工具, 以确定的机制, 减肥手术导致迅速和明显的改善肥胖并存。此外, 小鼠减肥手术模型提供了一个新的范式, 研究的基础生物学肠道调节各种过程, 如新陈代谢, 心血管功能, 并致癌<sup class="x...

随着肥胖流行病的持续增长, 世界范围的减肥手术越来越受欢迎, 因为它是最有效的长期治疗肥胖症的1。不幸的是, 节食和运动造成的减肥很难实现, 而且在长期的23中相对无效。减肥手术, 如垂直袖胃切除术 (VSG), 是指为减轻体重的目的而操作胃肠道1,4。虽然减肥是减肥手术的显著结果, 减肥手术提供了其他的健康好处, 如改善肥胖并存和延长寿命5。例如, 减肥手术导致2型糖尿病和高血压的高缓解率, 并减少某些类型的癌症的发展的终生风险1,6,7。值得注意的是, 减肥手术导致2型糖尿病和高血压缓解的效果往往是在手术后和体重减轻之前,8,9。这凸显了一个概念, 即有独立于体重的机制, 有助于在手术后观察到的健康益处。动物模型的减肥手术已经开发, 并被用来研究的机制, 这些健康的好处发生10,11,12。
我们已经验证了一个小鼠模型的 VSG, 我们已经应用于各种基因改良的小鼠模型, 以研究减肥手术改善肥胖并存, 如2型糖尿病, 高血压, 和结直肠癌的机制10,11,12. 啮齿类动物模型允许更多的实验控制和执行基因或药物操作的能力来定义特定基因的作用或信号通路的兴趣。我们主要关注 VSG, 因为 VSG 是美国临床上最常执行的减肥手术13。此外, VSG 是一个简单的手术模型, 较少解剖修改相比, 其他程序, 如糊-en-Y 胃旁路或胰分流。
我们的小鼠模型 VSG 重述的减肥手术观察到的以下影响: 体重下降, 减少食物摄入量, 改善葡萄糖调节, 改善胰岛功能, 增加 post-prandial 胰高血糖素样 peptide-1 (GLP-1) 分泌物,降低动脉血压, 增加循环胆汁酸浓度10,11,12,13,14,15。因此, 这是一个理想的模型来研究体重依赖性和独立的机制, VSG 改善或解决肥胖并存。此外, 它是一个可靠的模型, 可以结合其他手术程序, 允许调查的影响 VSG 在各种疾病的情况下与更大的机械洞察力12。

<table>
	<tbody>
		<tr>
			<td>45% high fat diet</td>
			<td>Research Diets</td>
			<td>D12451</td>
			<td></td>
		</tr>
		<tr>
			<td>60% high fat diet</td>
			<td>Research Diets</td>
			<td>D12492</td>
			<td></td>
		</tr>
		<tr>
			<td>Boost</td>
			<td>Nestle</td>
			<td>160-67538</td>
			<td>rich chocolate flavor</td>
		</tr>
		<tr>
			<td>6-0 Suture</td>
			<td>Ethicon</td>
			<td>Z432</td>
			<td>monofilament absorbable/taper</td>
		</tr>
		<tr>
			<td>7-0 Suture</td>
			<td>Covidien</td>
			<td>8866127-01</td>
			<td>monofilament absorbable/taper</td>
		</tr>
		<tr>
			<td>Cotton Swabs</td>
			<td>Fisherbrand</td>
			<td>23-400-118</td>
			<td>small</td>
		</tr>
		<tr>
			<td>Cotton Swabs</td>
			<td>Fisherbrand</td>
			<td>233-400-101</td>
			<td>large</td>
		</tr>
		<tr>
			<td>Gauze</td>
			<td>Various</td>
			<td></td>
			<td>4x4 4 ply and 2x2 4 ply</td>
		</tr>
		<tr>
			<td>Foil</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgery drape</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.9% saline solution</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LRS</td>
			<td>Hospira</td>
			<td>170RX</td>
			<td></td>
		</tr>
		<tr>
			<td>Betadine</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Eye Ointment</td>
			<td>Paralube&#174; Vet Ointment</td>
			<td>17033-211-38</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Adhesive</td>
			<td>Vetbond</td>
			<td>1469SB</td>
			<td></td>
		</tr>
		<tr>
			<td>Meloxicam (Metacam)</td>
			<td>Boehringer Ingelheim</td>
			<td>141-213</td>
			<td>5 mg/ml</td>
		</tr>
		<tr>
			<td>Enrofloxacin</td>
			<td>Baytril</td>
			<td>08713254-186599</td>
			<td>22.7 mg/ml</td>
		</tr>
		<tr>
			<td>Thin tipped hemostats</td>
			<td>Fine Science Tools</td>
			<td>13021-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Metzenbaum Scissor</td>
			<td>Fine Science Tools</td>
			<td>14018-18</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris Scissors</td>
			<td>Fine Science Tools</td>
			<td>14058-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont Forcep</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Serrated Forcep</td>
			<td>Fine Science Tools</td>
			<td>11020-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Gavage needle</td>
			<td>Fine Science Tools</td>
			<td>18060-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Microneedle driver</td>
			<td>Fine Science Tools</td>
			<td>12075-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Spring Scissor</td>
			<td>Fine Science Tools</td>
			<td>15396-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin syringe</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1mL syringe</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20mL syringe</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glucometer (one touch ultra mini)</td>
			<td>Lifescan</td>
			<td>70021208</td>
			<td></td>
		</tr>
		<tr>
			<td>Multiplex insulin and GLP-1 kit</td>
			<td>Meso Scale Discovery</td>
			<td>K15171C-1</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism 6.00</td>
			<td>GraphPad Software</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nestlets</td>
			<td>Ancare</td>
			<td>NES3600</td>
			<td></td>
		</tr>
	</tbody>
</table>

a murine model of vertical sleeve gastrectomy

减肥手术, 如垂直袖胃切除术 (VSG), 是一个手术的胃肠道, 是为了减肥的目的。减肥手术是目前最有效的长期治疗肥胖症。除了减肥, 减肥手术还会产生额外的健康好处, 如2型糖尿病缓解, 高血压缓解, 并减少罹患某些类型的癌症的风险。这些好处的减重机制仍未完全定义。因此, 动物模型的减肥手术正在开发和验证, 以确定机制, 导致这些好处, 目的是提高对胃肠生理的认识和确定新的治疗目标。VSG 已成为最常见的减肥手术在美国诊所, 因为它是非常有效的, 以产生体重下降和新陈代谢改善, 并比其他减肥程序更简单。因此, 我们已经开发和验证了一个小鼠模型的 VSG。这种小鼠 VSG 模型重述了许多 VSG 在人类中的作用, 其中包括改善葡萄糖和血压调节。该方法是根据胃的分离, 结扎胃血管, 并去除70% 的胃由 transecting 沿更大的曲率的胃。我们已成功地将这一手术协议应用于各种基因修饰的小鼠品系, 以确定机械贡献者对 VSG 的益处。此外, 这种小鼠 VSG 模型已与其他手术技术结合, 以获得更深的机械洞察力。因此, 这是一个简单而多用途的模型, 研究胃肠生理和减肥手术的健康效益。

在图 1中描述了假的和 VSG 的过程。图 1A显示缝合线在假手术过程中沿胃壁放置的位置。在 VSG 手术中, 这一区域是胃切开的地方。图 1B显示了 VSG 的表现后左胃的管状残留。
统计和数据分析 数据被显示为平均± SEM. 通过方差分析与 Tukey 的后测试?...

Watch this Scientific Journal Video about A Murine Model of Vertical Sleeve Gastrectomy at JoVE.com

A Murine Model of Vertical Sleeve Gastrectomy

下面介绍了垂直袖状胃切除术在小鼠中的表现。这是一种减肥手术, 包括切除约70% 的胃。

小鼠垂直套胃切除术的动物模型

Bariatric surgery, such as vertical sleeve gastrectomy (VSG), is a surgery of the gastrointestinal tract that is performed for the purpose of weight loss. ...

a-murine-model-of-vertical-sleeve-gastrectomy

Research

JoVE Journal

Biology

11.0K 次观看.  Cornell University. 下面介绍了垂直袖状胃切除术在小鼠中的表现。这是一种减肥手术, 包括切除约70% 的胃。

视频: A Murine Model of Vertical Sleeve Gastrectomy

Murine Pancreatic Islet Isolation

小鼠胰岛隔离

科研

生物学

Murine Model of Hindlimb Ischemia 

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide. Medical therapies for symptomatic relief are limited. Surgical or endovascular interventions are useful for some individuals, but long-term results are often disappointing. As a result, there is a need for developing new therapies to treat PAD. The murine hindlimb ischemia preparation is a model of PAD, and is useful for testing new therapies. When compared to other models of tissue ischemia such as coronary or cerebral artery ligation, femoral artery ligation provides for a simpler model of ischemic tissue. Other advantages of this model are the ease of access to the femoral artery and low mortality rate. 
In this video, we demonstrate the methodology for the murine model of unilateral hindimb ischemia. The specific materials and procedures for creating and evaluating the model will be described, including the assessment of limb perfusion by laser Doppler imaging. This protocol can also be utilized for the transplantation and non-invasive tracking of cells, which is demonstrated by Huang et al.1.

Murine Model of Hindlimb Ischemia

The surgical procedure for induction of unilateral hindlimb ischemia is demonstrated, with confirmation of ischemia by laser Doppler perfusion imaging.

肢体缺血小鼠模型

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide.  Medical therapies for ...

Murine Renal Transplantation Procedure

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell et al over 30 years ago (1) and refined by Zhang et al years later (2), few people in the world have mastered this procedure. In our laboratory we have successfully performed 1200 orthotopic kidney transplantations with &gt; 90% survival rate. The key points for success include stringent control of reperfusion injury, bleeding and thrombosis, both during the procedure and post-transplantation, and use of 10-0 instead of 11-0 suture for anastomoses.
Post-operative care and treatment of the recipient is extremely important to transplant success and evaluation. All renal graft recipients receive antibiotics in the form of an injection of penicillin immediately post-transplant and sulfatrim in the drinking water continually. Overall animal health is evaluated daily and whole blood creatinine analyses are performed routinely with a portable I-STAT machine to assess graft function.

Renal transplantation in mice is a technically challenging procedure that requires careful post-operative care and treatment for success.

小鼠肾移植程序

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell ...

LAD-Ligation: A Murine Model of Myocardial Infarction

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes in myocardial ischemia and to develop and optimize future treatment.
Two different methods of creating myocardial ischemia are performed in laboratory rodents. The first method is to create cryo infarction, a fast but inaccurate technique, where a cryo-pen is applied on the surface of the heart (1-3). Using this method the scientist can not guarantee that the cryo-scar leads to ischemia, also a vast myocardial injury is created that shows pathophysiological side effects that are not related to myocardial infarction. The second method is the permanent ligation of the left anterior descending artery (LAD). Here the LAD is ligated with one single stitch, forming an ischemia that can be seen almost immediately. By closing the LAD, no further blood flow is permitted in that area, while the surrounding myocardial tissue is nearly not affected. This surgical procedure imitates the pathobiological and pathophysiological aspects occurring in infarction-related myocardial ischemia.
The method introduced in this video demonstrates the surgical procedure of a mouse infarction model by ligating the LAD. This model is convenient for pathobiological and pathophysiological as well as immunobiological studies on cardiac infarction. The shown technique provides high accuracy and correlates well with histological sections.

This video demonstrates how to use a fast and reliable model to study pathobiological and pathophysiological processes of myocardial ischemia.

法援署结扎：有心肌梗死的小鼠模型

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

高效的生产和纯化重组小鼠Kindlin-3从昆虫细胞生物物理研究

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

鼠冠状动脉血管平滑肌细胞的分离

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

The Murine Choline-Deficient, Ethionine-Supplemented (CDE) Diet Model of Chronic Liver Injury

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual inflammation, progressive destruction and regeneration of the hepatic parenchyma, liver progenitor cell proliferation, and fibrosis. The end-stage of every chronic liver disease is cirrhosis, a major risk factor for the development of hepatocellular carcinoma. To study processes regulating disease initiation, establishment, and progression, several animal models are used in laboratories. Here we describe a six-week time course of the choline-deficient and ethionine-supplemented (CDE) mouse model, which involves feeding six-week old male C57BL/6J mice with choline-deficient chow and 0.15% DL-ethionine-supplemented drinking water. Monitoring of animal health and a typical body weight loss curve are explained. The protocol demonstrates the gross examination of a CDE-treated liver and blood collection by cardiac puncture for subsequent serum analyses. Next, the liver perfusion technique and collection of different hepatic lobes for standard evaluations are shown, including liver histology assessments by hematoxylin and eosin or Sirius Red stainings, immunofluorescent detection of hepatic cell populations as well as transcriptome profiling of the liver microenvironment. This mouse model is suitable for studying inflammatory, fibrogenic, and liver progenitor cell dynamics induced through chronic liver disease and can be used to test potential therapeutic agents that may modulate these processes.

The Murine Choline-Deficient, Ethionine-Supplemented CDE Diet Model of Chronic Liver Injury

Here we describe a common method to induce chronic liver injury in mice by feeding of a choline-deficient and ethionine-supplemented (CDE) diet. We demonstrate health monitoring, liver perfusion, isolation, and preservation. A time course of six weeks can inform about liver injury, pathohistology, fibrosis, inflammatory, and liver progenitor cell responses.

慢性肝损伤小鼠胆碱缺乏、氨基补充的饮食模型

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

质谱分析，用于识别 EYFP 标记 CENP-A （EYFP-CENP-A） 的泛化

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

A Murine Model of a Burn Wound Reconstructed with an Allogeneic Skin Graft

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and require surgical debridement and skin grafting. Successful integration of the donor graft into a recipient wound bed depends on timely recruitment of immune cells, robust angiogenic response and new extracellular matrix formation. The development of novel therapeutic agents, which target some key processes involved in wound healing, are hindered by the lack of reliable preclinical models with optimized objective assessment of wound closure. Here, we describe an inexpensive and reproducible model of experimental full thickness burn wound reconstructed with an allogeneic skin graft. The wound is induced on the dorsum surface of anaesthetized inbred wild type mice from the BALB/C and SKH1-Hrhr backgrounds. The burn is produced using a brass template measuring 10 mm in diameter, which is preheated to 80 &#176;C and delivered at a constant pressure for 20 s. Burn eschar is excised 24 hours after the injury and replaced with a full thickness graft harvested from the tail of a genetically similar donor mouse. No specialized equipment is required for the procedure and surgical techniques are straightforward to follow. The method may be effortlessly implemented and reproduced in most research settings. Certain limitations are associated with the model. Due to technical difficulties, the harvest of thinner split thickness skin grafts is not possible. The surgical method we describe here allows for the reconstruction of burn wounds using full thickness skin grafts. It may be used to carry out preclinical therapeutic testing.

The aim of this study was to develop a murine model of burn wound healing. A thermal burn was induced on the dorsal skin of mice using a preheated brass template. Burned tissue was debrided and overlaid with a skin graft harvested from the tail of a genetically similar donor mouse.

用异体皮肤移植重建的烧伤伤口的穆林模型

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and ...

Comprehensive Echocardiographic Assessment of Right Ventricle Function in a Rat Model of Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a progressive disease caused by vasoconstriction and remodeling of the small arteries in the lungs. This remodeling leads to increased pulmonary vascular resistance, worsened right ventricular function, and premature death. Currently approved therapies for PAH largely target pulmonary vasodilator pathways; however, recent emerging therapeutic modalities are focused on other novel pathways involved in the pathogenesis of the disease, including right ventricle (RV) remodeling. Imaging techniques that allow longitudinal assessment of novel therapeutics are very useful for determining the efficacy of new drugs in preclinical studies. Noninvasive trans-thoracic echocardiography remains the standard approach to evaluating heart function and is widely used in rodent models. However, echocardiographic evaluation of the RV can be challenging due to its anatomical position and structure. In addition, standardized guidelines are lacking for echocardiography&#160;in preclinical rodent models, making it difficult to carry out a uniform assessment of RV&#160;function across studies in different laboratories. In preclinical studies, the monocrotaline (MCT) injury model in rats is widely used to evaluate drug efficacy for treating PAH. This protocol describes the echocardiographic evaluation of the RV in na&#239;ve and MCT-induced PAH rats.

The present protocol describes the echocardiographic characterization of right ventricular morphology and function in a rat model of pulmonary arterial hypertension.