笔者想感谢好心J.路德博士和K. Ubieta准备好数据和BGrötsch博士校对稿件。这项工作是由德意志研究联合会（BO3811 / 1-1-埃米诺特）的支持。

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脂肪细胞是由它们的尺寸，数量和面积表型特征，揭示脂肪细胞增生和肥大，由过多的能量存储诱导由于过度营养5。这些事件导致脂肪酸失调和随后的代谢综​​合征也与保存的代谢，其也被称为"健康的"脂肪扩张规定增加脂肪量。例如，Kusminski 等人 21表明，大规模的扩张胖老鼠保持健康的新陈代谢，表明脂肪扩张不一定与代谢综合征，需要慎重考虑来评估脂肪细胞的特性。脂肪组织（AT）起着机体代谢的调节了举足轻重的作用。 AT是最大的内分泌器官可能影响血脂异常，动脉粥样硬化，高胰岛素血症和高血糖3。评估AT稳态和分子米echanisms调节它可以允许更好地理解代谢系统疾病。因此，解开调节脂肪细胞分化机制，脂肪细胞的大小和脂肪垫质量，将有助于开发肥胖疾病新的治疗治疗。 使用体内 和体外方法，它能够确定的食物和基因表达的影响对脂肪细胞分化和活性的作用。来确定所述AT稳态，确定脂肪细胞分化，增殖和凋亡之间的平衡的建议由我们的协议是作为分析葡萄糖和胰岛素代谢反应22-24一样重要。 参与脂肪形成，脂肪生成，脂肪分解，脂肪酸的摄取，缺氧，细胞凋亡与增殖的主要脂肪细胞基因的表达谱分析和内脏AT是获得对脂肪细胞稳态和可能的功能障碍的概述的高通量方法。有趣的考生应在通过免疫印迹或免疫组织化学染色的蛋白水平进行进一步分析。使用非对称的花青染料获得通过实时PCR系统获得最佳效果，cDNA的取值范围为1至10毫微克的浓度和最佳引物浓度范围为50至900纳米应当进行测试，以最小化非特异性扩增。关键组件是引物;对于每次运行，熔解曲线需要进行严格的控制，以确保特异性和排除引物二聚体的形成。因此，建议使用用H 2 O代替cDNA的阴性对照。此外，市售不对称菁染料是作为包含一个被动参比染料（如ROX），以提供内部参考信号预混液。的cDNA的信号数据分析的ROX信号期间归纠正小康的好信号波动。另一点，以建立一个咕考虑ð定量PCR系统是看家基因的选择。对于各条件，几个持家基因的使用， 例如，HPRT，β 肌动蛋白 ，GAPDH，β-2微球蛋白或HSP90。 通过在低氧条件稳定HIF蛋白是主调节器确定不仅脂肪细胞的生存，但也代谢改变，如葡萄糖，胰岛素耐受性和脂质代谢11,25,26。为了确定在脂肪垫缺氧地区，HIF-1α是通过免疫组化的部分来确定英寸由于HIF蛋白含氧量正常条件下5至10分钟内迅速降解，该过程和脂肪垫的用于组织学分析的固定应严格控制，以避免等待时间。因此，不仅通过HIF染色确保缺氧，pimonidazole用于确定在AT缺氧区域。 Pimonidazole能够分配到组织中，因为它是在骨头27已经示出和有效地通过结合到特异性在低氧细胞含硫醇的蛋白质，其进一步在组织切片由特异性抗体结合28检测标记缺氧区域。然而，其他的标记物和方法可用于分析缺氧通路。例如，脯氨酰羟化酶（PHD）的酶，从而诱发在常氧脯氨酸残基的羟基化，以及希佩尔 - 林道（VHL）蛋白，其识别羟基脯氨酸和诱导多聚泛素介导的蛋白酶体HIF的参与退化，需要分析的通路29,30的完整概述。此外，HIFs无所不在检测也将确定蛋白质的稳定性和降解，可以改变31,32。 此外，通过Ki67的增殖和凋亡TUNEL染色在体内通过AT组织切片染色确定。通过Ki67和增殖的量化凋亡的影响TUNEL法或膜联蛋白V染色，通过流式细胞仪分析，也进行了33。增殖当然可以通过其它技术，如脂肪细胞的细胞周期，这是不通过的Ki67阳性细胞的测定处理物的分析测定。而且，可以完成用TUNEL凋亡研究膜联蛋白V和TOP-PR-3，其将确定坏死与细胞凋亡的细胞死亡过程的水平通过FACS分析。凋亡是细胞死亡的方案，这是对于AT稳态重要的基本过程。事实上，脂肪细胞凋亡的失调先前已在促进肥胖过程牵连脂肪代谢障碍34。此外，在2011年，Keuper 等挂钩脂肪组织炎症脂肪细胞凋亡。它们表明，巨噬细胞诱导的细胞凋亡中的前脂肪细胞和脂肪细胞，这反过来吸引巨噬细胞。巨噬细胞的招募加速炎症，从而对照ibutes代谢综合征例如葡萄糖和胰岛素耐受性35。然而，脂肪细胞凋亡仍然是一个研究的不良现象，尽管假设，即诱导脂肪细胞的凋亡可导致降低的重量。 本协议采用免疫组化方法来研究不同的现象，如在体内增殖，凋亡和缺氧。因此，将组织用4％的甲醛，这是一个关键步骤。延长组织固定时间导致改变的表位，这成为抗体不可访问的。相反，短的固定时间增加表位的给试剂的敏感性。固定的推荐最佳时间为24小时。此外，该部分的厚度也影响抗体结合到其表位;最佳厚度为2和5微米之间。部分超过5微米厚会由于增加的结合位点得到假阳性结果。相比之下，Sections薄于2μm的含有较少的结合位点和正面的区域没有得到很好的限定。进一步关键因素是所述抗体本身，孵育时间，浓度和温度均匀，这影响特异性结合的表位的质量。因此，验证抗体浓度和温育时间是必要的各条件。 为完成此研究中，我们提供了一种在体外脂肪细胞分化的协议，这可能是由不同的处理，刺激或共培养进行扩展。通过使用体外脂肪细胞培养物，它是可行的，以确定在脂肪细胞分化和功能的缺陷。为了获得可靠的结果，为所有原代细胞，脂肪干细胞的脂肪细胞和的健康行为和外观是非常重要的。粒度，细胞质vacuolations和/或脱离是恶化的迹象，表明不足介质，微生物污染或初级的衰老细胞。此协议是使用分离的脂肪细胞从脂肪垫组织，而其是也可以使用由其它协议36描述从骨髓中分离间充质干细胞。最新的包括间质祖细胞，这可能反映了脂肪细胞分化的非常早一步发生的额外分化的问题，这可能会在我们目前的协议被错过。此外，脂肪干细胞能够迅速地（在一周内的10倍以上）扩大，并且有些段落后长期培养的脂肪干细胞仍保持它们间质多能性37,38。使用脂肪干细胞的另一个优点是，可以很容易地切换到人体中，由于脂肪干细胞可以从患者通过吸脂这是一个简单的微创的方法进行收获。 作为以内分泌方式影响等几个器官，该协议应延长至脂肪细胞因子。脂肪因子，如瘦素，脂联素，肿瘤坏死因子α（TNF）一第二抵抗，脂肪细胞分泌的已知通过控制脂肪代谢，能量平衡和胰岛素敏感性39，影响代谢性疾病。因此，血清和脂肪细胞分泌组分析应该执行。在AT功能障碍，脂肪因子和促炎细胞因子，如IL-6的情况下，可导致器官失调如肝脏和胰腺和肌肉功能4。为了排除全身器官功能障碍，动物模型或细胞培养物可为他们的反应对葡萄糖刺激和摄取进行测试。 在这里，我们分析了AT和体内脂肪细胞的基本状态提供的协议并在体外以显示脂肪细胞内稳态和功能的分子机制。

据世界卫生组织2014年的报告显示，全球成人人口的39％超重，13％肥胖1。在不久的将来，超重的人将包括老年人口的比例显著。肥胖和老化的一个重要特征是相对于发病率和死亡率2的脂肪失调。脂肪因子，由脂肪组织（AT）的分泌的蛋白质，可以触发代谢综合征如肥胖症和2型糖尿病3。代谢性疾病主要是由过多的能量储存在脂肪细胞的脂滴，这导致在扩张4引起的。因此，感兴趣的确定，以便找到的机会，以控制它的原因和AT膨胀的分子机制。 营养过剩导致的AT扩张，这是由两个事件调节:过量的能量存储为脂肪细胞的脂滴，一个处理导致肥大（在脂肪细胞尺寸增加），并增加了脂肪生成，也称为脂肪细胞增生5。脂肪生成是多能间充质干细胞（MSC）为脂肪细胞的分化的方法。首先，干细胞在承诺阶段发展成脂肪前体细胞。其次，前脂肪细胞进一步分化获得成熟和功能的脂肪细胞6的功能。一些转录因子已被确定为主调节器前脂肪细胞测定，如锌指蛋白423（Zfp423）和早期B细胞因子1（EBF1）。而Zfp423诱导早承诺，EBF1需要脂肪细胞祖细胞6的产生。终末分化紧密由转录级联，由此过氧化物酶体增殖物激活受体γ（PPARγ）是必需的转录因子7控制。进一步关键转录因子是CCAAT /增强子结合蛋白（C/ EBP）家族成员（ 即 C /EBPα，C /EBPβ和C /EBPδ），Kruppel样因子（KLFs），cAMP反应元件结合蛋白（CREB）和早期生长反应20（猪Krox20）6。 最近，已经表明，激活蛋白-1（AP-1）家族参与在脂肪细胞分化过程8,9。该AP-1系列由二聚体蛋白复合物，低聚果糖，君和/或激活转录因子（ATF）成员组成的形成。 Fos蛋白相关抗原1和2（FRA-1和FRA-2）是能调节脂肪细胞分化。 FRA-1通过抑制C /EBPα8，而FRA-2控制脂肪细胞的营业额9削弱脂肪细胞分化。离-2-从而不仅由脂肪细胞分化过程中抑制PPARγ2表达降低了脂肪细胞数目，而且还通过缺氧诱导因子（HIFs）的表达的直接压制降低脂肪细胞的细胞凋亡。该HIF家庭是一个烯酸rodimeric转录因子复合物，HIF-1α，HIF-2α和HIF-1β组成。该异二聚体由氧敏感的HIF-α蛋白（HIF-1α或HIF-2α）和氧气不敏感的HIF-1β亚单位10。在常氧，HIF-α蛋白聚ubiquitinylated并最终被蛋白酶体降解11。在缺氧条件下，在膨胀过程中的AT发生，HIF-α蛋白是不再羟基化。因此，他们成为稳定并形成二聚体与组成性表达HIF-1β。由HIF应答元件控制的基因的转录激活是参与血管发生，代谢和炎症12的调节。事实上，HIF-1α诱导的葡萄糖耐受性，抑制能量消耗和外围使用脂质，以及通过增加瘦素水平与HFD诱导的肝脂肪变性13促进AT功能障碍。此外，HIF-1α的调节adipocy德凋亡的体内 和体外 9。 本协议描述为就读于状态解开在成年小鼠脂肪细胞稳态的分子特征的方法。它显示了如何凋亡，增殖和体内 和体外脂肪细胞的分化可以通过缺氧调节。要做到这一点，我们使用与携带渡与FABP4-CreERT小鼠9 FRA-2等位基因两侧装接loxP小鼠产生FRA-2的脂肪细胞特异性缺失的小鼠。通过使用FABP4 Cre重组酶的小鼠ERT，删除是脂肪细胞特异性和诱导他莫昔芬注射14。对于成人模型，他莫昔芬的腹膜内注射在连续5天开始至6周的年龄进行。因此，所做的分析之前将小鼠进行正常饮食或高脂肪饮食6周。在此研究中使用的小鼠基于C57BL6背景雄性避免雌激素，如雌激素，显示调节身体脂肪的分布15。使用另一种遗传背景也可能改变的代谢表型，由于在脂质管理16应变相关的差异。 此协议演示了如何使用组织学分析AT缺氧条件下，如何利用免疫组织化学和基因分析分析，以量化的脂肪细胞凋亡，增殖和分化的体内 。这项研究是由体外实验，展示如何分析主要脂肪细胞分化和凋亡暴露在缺氧的改变完成。

<table>
	<tbody>
		<tr>
			<td>RNAlater solution</td>
			<td>Ambion</td>
			<td>AM7021</td>
			<td>RNA stabilization solution</td>
		</tr>
		<tr>
			<td>High-Capacity cDNA Reverse Transcription Kit</td>
			<td rowspan="2">Applied Biosystems</td>
			<td>4368813</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Select Master Mix</td>
			<td>4472908</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified Mouse Anti-Human Ki-67&#160;&#160;</td>
			<td rowspan="2">BD Biosciences</td>
			<td>550609</td>
			<td>Clone &#160;B56&#160;(RUO)</td>
		</tr>
		<tr>
			<td>Purified Mouse Anti-Human Ki-67&#160; Clone &#160;B56&#160;(RUO)</td>
			<td>550609</td>
			<td>Proliferation marker</td>
		</tr>
		<tr>
			<td>FITC Annexin V</td>
			<td>BioLegend</td>
			<td>640906</td>
			<td></td>
		</tr>
		<tr>
			<td>Cleaved Caspase-3 Rabbit mAb</td>
			<td rowspan="2">Cell signalling</td>
			<td>9664S</td>
			<td>&#160;Clone 5A1E</td>
		</tr>
		<tr>
			<td>Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb</td>
			<td>9664</td>
			<td>Apoptosis marker</td>
		</tr>
		<tr>
			<td>Hypoxyprobe-1 Plus Kit</td>
			<td>Hypoxyprobe</td>
			<td>HP2-200Kit</td>
			<td>Hypoxia marker, Solid pimonidazole HCl (Hypoxyprobe-1), FITC conjugated to mouse IgG&#8321; monoclonal antibody (FITC-MAb1), Rabbit anti-FITC conjugated with horseradish peroxidase</td>
		</tr>
		<tr>
			<td>Lipofectamine2000 Reagent&#160;</td>
			<td rowspan="2">Invitrogen</td>
			<td>11668-027</td>
			<td></td>
		</tr>
		<tr>
			<td>TO-PRO-3 Iodide</td>
			<td>T3605</td>
			<td>Nuclear counterstain, Monomeric cyanine nucleic acid stain, Excitation&#8260;Emission: 642&#8260;661 nm</td>
		</tr>
		<tr>
			<td>Mayer&#8217;s hemalum</td>
			<td>Merck</td>
			<td>109249</td>
			<td>hematoxylin</td>
		</tr>
		<tr>
			<td>pegGOLD TriFast</td>
			<td rowspan="3">Peqlab</td>
			<td>30-2030</td>
			<td>TRIzol, single-phase solution of guanidinisothiocyanat and phenol</td>
		</tr>
		<tr>
			<td>Percellys Ceramic Kit 1.4 mm</td>
			<td>91-PCS-CK14</td>
			<td>tubes containing ceramic beads (1.4 mm)</td>
		</tr>
		<tr>
			<td>Precellys 24</td>
			<td>91-PCS24</td>
			<td>homogenizer</td>
		</tr>
		<tr>
			<td>HIF-1 alpha Antibody</td>
			<td rowspan="2">Pierce</td>
			<td>PA1-16601</td>
			<td></td>
		</tr>
		<tr>
			<td>HIF-1 alpha Antibody, 16H4L13</td>
			<td>700505</td>
			<td>Hypoxia marker</td>
		</tr>
		<tr>
			<td>In Situ Cell Death Detection Kit, Fluorescein</td>
			<td>Roche</td>
			<td>11 684 795 001</td>
			<td>TdT-mediated dUTP-biotin nick end labeling (TUNEL)&#160;</td>
		</tr>
		<tr>
			<td>Eosin</td>
			<td>Sigma</td>
			<td>318906</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I Solution (1 unit/&#181;L)</td>
			<td>Thermo Scientific</td>
			<td>EN0525</td>
			<td></td>
		</tr>
		<tr>
			<td>biotinylated anti mouse IgG (H+L)</td>
			<td rowspan="4">Vector Laboratories</td>
			<td>BA-9200</td>
			<td></td>
		</tr>
		<tr>
			<td>biotinylated anti mouse IgG (H+L)</td>
			<td>BA-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectastain ABC Kit</td>
			<td>PK-4000&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>VECTASHIELD Mounting Medium with DAPI</td>
			<td>H-1200</td>
			<td></td>
		</tr>
	</tbody>
</table>

mechanism of regulation of adipocyte numbers in adult organisms through differentiation and apoptosis homeostasis

Considering that adipose tissue (AT) is an endocrine organ, it can influence whole body metabolism. Excessive energy storage leads to the dysregulation of adipocytes, which in turn induces abnormal secretion of adipokines, triggering metabolic syndromes such as obesity, dyslipidemia, hyperglycemia, hyperinsulinemia, insulin resistance and type 2 diabetes. Therefore, investigating the molecular mechanisms behind adipocyte dysregulation could help to develop novel therapeutic strategies. Our protocol describes methods for evaluating the molecular mechanism affected by hypoxic conditions of the AT, which correlates with adipocyte apoptosis in adult mice. This protocol describes how to analyze AT in vivo through gene expression profiling as well as histological analysis of adipocyte differentiation, proliferation and apoptosis during hypoxia exposure, ascertained through staining of hypoxic cells or HIF-1&#945; protein. Furthermore, in vitro analysis of adipocyte differentiation and its responses to various stimuli completes the characterization of the molecular pathways behind possible adipocyte dysfunction leading to metabolic syndromes.

我们显示如何使用的实例，以确定在体内和体外的脂肪细胞内稳态FRA-2 FL / FL与野生型同窝FABP4-CreERT小鼠。我们的协议定义了如何提高脂肪细胞的凋亡指示缺氧增加HIF表达与脂肪细胞的功能障碍有关。 增加脂肪细胞尺寸和面积在高脂肪饮食（HFD）的处理的小鼠营养过剩，除其他因素外，导致脂肪细胞肥大引起的过多的能量存储在脂滴。脂肪细胞的尺寸和面积是肥大的指标。从正常的脂肪垫的区段（ND; 图6a）和高脂肪饮食（HFD; 图6b）小鼠以及脂肪细胞的尺寸和面积的quantifications清楚显示脂肪细胞6周后肥大HFD，第这是由增加的脂肪细胞大小在HFD处理的小鼠（ 图6c和d）来表示。 在成人的脂肪组织（AT）的上升缺氧FRA-2 FL / FL FABP4-CreERT小鼠导致增加HIF-1α的水平和脂肪细胞的凋亡以确定在AT缺氧的体内状态，FRA-2 FL / FL FABP4-CreERT小鼠在12周龄FRA-2缺失6周后进行分析，并与野生型同窝相比。 Pimonidazole施用给小鼠腹膜内作为有效缺氧标记;它是无毒的，并且能够分配到所述AT。在所述AT缺氧脂肪细胞在体内被免疫抗体染色（ 图7a）限定。此外，在小鼠中的AT的增加的缺氧状态是伴随着增加HIF-1α阳性ADIP通过免疫组织化学染色图7b），这是由HIF-1α的表达水平和其靶基因9的定量确认所指示ocytes。此外，从FRA-2的AT切片的TUNEL染色FL / FL FABP4-CreERT小鼠和对照同窝（ 图7c）示出了增加的脂肪细胞的凋亡与缺氧和HIF-1α表达的存在相关。 通过缺氧诱导的脂肪细胞凋亡增加初级脂肪细胞HIF-1α表达分析脂肪细胞凋亡作用 ，我们使用来自皮下脂肪垫脂肪细胞产生的其他地方20所描述。正如所料，在脂肪细胞中的HIF-1α表达后缺氧（ 图8a）中的24小时的增加。进一步分析，HIF-1α的活动，HIF靶的RNA水平基因如iNOS基因蛋白（诱导型一氧化氮合酶）通过qPCR定量。 图8B显示了HIF-1α的低氧条件下的表达增加导致增加的iNOS mRNA水平的水平。因为我们已经在体内 （ 图8），在脂肪细胞增加HIF-1α的表达具有增加的脂肪细胞的凋亡相关已经示出，凋亡也在体外培养物通过膜联蛋白V染色低氧条件下进行定量。 与体内数据（ 图7）相一致，增加HIF-1α的水平是伴随着增加的脂肪细胞的细胞凋亡诱导的缺氧条件下（ 图8b）。此外，为了证明缺氧诱导的细胞凋亡是HIF依赖性; HIF-1α或HIF-2α通过RNA干扰在从野生型或FRA-2缺陷型小鼠的脂肪细胞沉默。增加的脂肪细胞的细胞凋亡是由沉默HIF-1α或HIF-2α恢复如由膜联蛋白<html> V染色在图8b中，证明了低氧传感器HIF-α调节脂肪细胞的细胞凋亡。 <img alt="图6" src="/files/ftp_upload/53822/53822fig6.jpg" /> 图6:提高在高脂肪饮食（HFD）的小鼠脂肪细胞尺寸和面积从与正常饮食（ND）供给雄性野生型小鼠的perigonadal脂肪垫部（A，B）的H＆E染色（a）或高。 -fat饮食（HFD）（b）为6周。条代表500微米。 （C，D）从具有ND（a）或高脂饮食喂养6周（B）野生型小鼠的perigonadal脂肪垫的脂肪细胞大小（c）和区域（d）的Quantifications。每组10数据显示为平均值±SEM表示。使用Student&#39;s t检验进行统计分析。 *** P &lt;0.0001。://www.jove.com/files/ftp_upload/53822/53822fig6large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图7" src="/files/ftp_upload/53822/53822fig7.jpg" /> 图7:增加HIF-1α水平和凋亡的FRA-2 FL / FL FABP4-CreERT 小鼠 成年脂肪细胞 。缺氧（a）和HIF-1α（b）以雄性FRA-2 FL / FL 6周后FABP4-creERT小鼠和雄性对照同窝三苯氧胺喷射的AT染色。放大倍数20X，40X插入。黑色箭头指示缺氧区和HIF-1α阳性的细胞。 （ 三 ）TUNEL法FRA-2 FL / FL FABP4-CreERT小鼠和控制同窝AT他莫昔芬注射后6周。放大倍数20X，40X插入。黑色箭头表示TUNEL阳性细胞。这个数字已经从路德等修饰人9。 <a href="https://www.jove.com/files/ftp_upload/53822/53822fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图8" src="/files/ftp_upload/53822/53822fig8.jpg" /> 图8: 增加HIF-1α表达和脂肪细胞在缺氧诱导初级脂肪细胞的凋亡 （ 一 ）HIF-1α和HIFs目标中iNOS mRNA水平放置在缺氧室原脂肪细胞的实时PCR分析来分析在指定的时间点。 （b）由在从分离的原代脂肪细胞膜联蛋白V FACS染色的细胞凋亡的定量FRA-2 FL / FL与SH对照或抗HIF-1α或HIF-2α的sh质粒转染和缺氧条件下放置FABP4-CreERT小鼠或野生型对照（1％O 2），24小时。这个数字已经从路德等修饰。 <sup&gt; 9。数据示±用斯氏t检验进行的SD统计分析作为平均值。 * P &lt;0.05，** P &lt;0.01被接受为显著。 <a href="https://www.jove.com/files/ftp_upload/53822/53822fig8large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Mechanism of Regulation of Adipocyte Numbers in Adult Organisms Through Differentiation and Apoptosis Homeostasis at JoVE.com

Mechanism of Regulation of Adipocyte Numbers in Adult Organisms Through Differentiation and Apoptosis Homeostasis

Adipose tissue (AT) can influence whole body homeostasis, therefore understanding the molecular mechanisms of adipocyte differentiation and function is of importance. We provide a protocol for gaining new insights into these processes by analyzing adipocyte homeostasis, differentiation and hypoxia exposure as a model for induced adipocyte apoptosis.

成人脂肪细胞生物体的数字调控机制，通过分化与凋亡的动态平衡

Considering that adipose tissue (AT) is an endocrine organ, it can influence whole body metabolism. Excessive energy storage leads to the dysregulation of ...

mechanism-regulation-adipocyte-numbers-adult-organisms-through

Research

JoVE Journal

Developmental Biology

14.9K 次观看.  Universitätsklinikum Erlangen. Adipose tissue (AT) can influence whole body homeostasis, therefore understanding the molecular mechanisms of adipocyte differentiation and function is of importance. We provide a protocol for gaining new insights into these processes by analyzing adipocyte homeostasis, differentiation and hypoxia exposure as a model for induced adipocyte apoptosis.

视频: Mechanism of Regulation of Adipocyte Numbers in Adult Organisms Through Differentiation and Apoptosis Homeostasis

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

科研

发育生物学

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated by transcription factors which bind genomic regulatory regions including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to chemical modifications. Modifications of histones further lead to repressed, activated or poised gene transcription, thus bringing another level of fine tuning regulation of gene transcription. Embryonic Stem cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development. They provide in principle enough material for chromatin immunoprecipitation (ChIP), a technology broadly used to identify gene regulatory regions. Furthermore, human ES cells represent a human cell model of cardiogenesis. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or ChIP-sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription.

A fine tuning regulation of gene transcription underlies embryonic cell fate decision. Herein, we describe chromatin immunoprecipitation assays used to investigate epigenetic regulation of both cardiac differentiation of stem cells and cardiac development of mouse embryos.

心脏分化的表观遗传调控胚胎干细胞和组织

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

Isolating Hair Follicle Stem Cells and Epidermal Keratinocytes from Dorsal Mouse Skin

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by distinct pools of SCs, which are located in the permanent portion of the HF, a region known as the bulge. These multipotent bulge SCs were initially identified as slow cycling label retaining cells; however, their isolation has been made feasible after identification of specific cell markers, such as CD34 and keratin 15 (K15). Here, we describe a robust method for isolating bulge SCs and epidermal keratinocytes from mouse HFs utilizing fluorescence activated cell-sorting (FACS) technology. Isolated hair follicle SCs (HFSCs) can be utilized in various in vivo grafting models and are a valuable in vitro model for studying the mechanisms that govern multipotency, quiescence and activation.

An ideal model for studying adult stem cell biology is the mouse hair follicle. Here we present a protocol for isolating different populations of hair follicles stem cells and epidermal keratinocytes, employing enzymatic digestion of mouse dorsal skin followed by FACS analysis.

从背小鼠皮肤分离毛囊干细胞和表皮角质形成细胞

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

人多能干细胞对原始和最终造血祖细胞定向分化的研究

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, antibodies for specific cell markers are applied on tissue sections. In adult skeletal muscle, satellite cells (SCs) are stem cells that contribute to muscle repair and regeneration. Therefore, it is important to visualize and trace the satellite cell population under different physiological conditions. In resting skeletal muscle, SCs reside between the basal lamina and myofiber plasma membrane. A commonly used marker for identifying SCs on the myofibers or in cell culture is the paired box protein Pax7. In this article, an optimized Pax7 immunofluorescence protocol on skeletal muscle sections is presented that minimizes non-specific staining and background. Another antibody that recognizes a protein (laminin) of the basal lamina was also added to help identify SCs. Similar protocols can also be used to perform double or triple labeling with Pax7 and antibodies for additional proteins of interest.

The precise identification of satellite cells is essential for studying their functions under various physiological and pathological conditions. This article presents a protocol to identify satellite cells on adult skeletal muscle sections by immunofluorescence-based staining.

Pax7 和层粘连蛋白抗体免疫荧光法鉴定骨骼肌卫星细胞

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, ...

Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies have demonstrated the wide differentiation capacity that these cells possess. The ovary represents a promising candidate for cell-based therapies due to the fact that it is rich in MSCs and that it is frequently discarded after ovariectomy surgeries as biological waste. This article describes procedures for the isolation, expansion, and differentiation of MSCs derived from the canine ovary, without the necessity of cell-sorting techniques. This protocol represents an important tool for regenerative medicine because of the broad applicability of these highly differentiable cells in clinical trials and therapeutic uses.

Herein, we describe a method for the isolation, expansion, and differentiation of mesenchymal stem cells from canine ovarian tissue.

犬卵巢间充质干细胞的提取与分化

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies ...

Visualize Drosophila Leg Motor Neuron Axons Through the Adult Cuticle

The majority of work on the neuronal specification has been carried out in genetically and physiologically tractable models such as C. elegans, Drosophila larvae, and fish, which all engage in undulatory movements (like crawling or swimming) as their primary mode of locomotion. However, a more sophisticated understanding of the individual motor neuron (MN) specification&#8212;at least in terms of informing disease therapies&#8212;demands an equally tractable system that better models the complex appendage-based locomotion schemes of vertebrates. The adult Drosophila locomotor system in charge of walking meets all of these criteria with ease, since in this model it is possible to study the specification of a small number of easily distinguished leg MNs (approximately 50 MNs per leg) both using a vast array of powerful genetic tools, and in the physiological context of an appendage-based locomotion scheme. Here we describe a protocol to visualize the leg muscle innervation in an adult fly.

Visualize Drosophila Leg Motor Neuron Axons Through the Adult Cuticle

Here we describe a protocol to visualize the axonal targeting with a florescent protein in adult legs of Drosophila by fixation, mounting, imaging, and post-imaging steps.

通过成人角质层可视化果蝇腿运动神经元轴突

The majority of work on the neuronal specification has been carried out in genetically and physiologically tractable models such as C. elegans, Drosophila ...

Induction of Endothelial Differentiation in Cardiac Progenitor Cells Under Low Serum Conditions

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are extremely scarce, but expanded CPCs could be useful for cell therapy. A prerequisite for their use is their ability to differentiate in a controlled manner into the various cardiac lineages using defined and efficient protocols. In addition, upon in vitro expansion, CPCs isolated from patients or preclinical disease models may offer fruitful research tools for the investigation of disease mechanisms.
Current studies use different markers to identify CPCs. However, not all of them are expressed in humans, which limits the translational impact of some preclinical studies. Differentiation protocols that are applicable irrespective of the isolation technique and marker expression will allow for the standardized expansion and priming of CPCs for cell therapy purpose. Here we describe that the priming of CPCs under a low fetal bovine serum (FBS) concentration and low cell density conditions facilitates the endothelial differentiation of CPCs. Using two different subpopulations of mouse and rat CPCs, we show that laminin is a more suitable substrate than fibronectin for this purpose under the following protocol: after culturing for 2 - 3 days in medium including supplements that maintain multipotency and with 3.5% FBS, CPCs are seeded on laminin at &#60;60% confluence and cultured in supplement-free medium with low concentrations of FBS (0.1%) for 20 - 24 hours before differentiation in endothelial differentiation medium. Because CPCs are a heterogeneous population, serum concentrations and incubation times may need to be adjusted depending on the properties of the respective CPC subpopulation. Considering this, the technique can be applied to other types of CPCs as well and provides a useful method to investigate the potential and mechanisms of differentiation and how they are affected by disease when using CPCs isolated from respective disease models.

This protocol describes an endothelial differentiation technique for cardiac progenitor cells. It particularly focuses on how serum concentration and cell-seeding density affect the endothelial differentiation potential.

低血清条件下心脏祖细胞内皮分化的诱导作用

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are ...

Accurate Follicle Enumeration in Adult Mouse Ovaries

Sexually reproducing female mammals are born with their entire lifetime supply of oocytes. Immature, quiescent oocytes are found within primordial follicles, the storage unit of the female germline. They are non-renewable, thus their number at birth and subsequent rate of loss largely dictates the female fertile lifespan. Accurate quantification of primordial follicle numbers in women and animals is essential for determining the impact of medicines and toxicants on the ovarian reserve. It is also necessary for evaluating the need for, and success of, existing and emerging fertility preservation techniques. Currently, no methods exist to accurately measure the number of primordial follicles comprising the ovarian reserve in women. Furthermore, obtaining ovarian tissue from large animals or endangered species for experimentation is often not feasible. Thus, mice have become an essential model for such studies, and the ability to evaluate primordial follicle numbers in whole mouse ovaries is a critical tool. However, reports of absolute follicle numbers in mouse ovaries in the literature are highly variable, making it difficult to compare and/or replicate data. This is due to a number of factors including strain, age, treatment groups, as well as technical differences in the methods of counting employed. In this article, we provide a step-by-step instructional guide for preparing histological sections and counting primordial follicles in mouse ovaries using two different methods: [1] stereology, which relies on the fractionator/optical dissector technique; and [2] the direct count technique. Some of the key advantages and drawbacks of each method will be highlighted so that reproducibility can be improved in the field and to enable researchers to select the most appropriate method for their studies.

Here, we describe, compare, and contrast two different techniques for accurate follicle counting of fixed mouse ovarian tissues.