Name: non-invasive ultrasound assessment of endometrial cancer progression in pax8-directed deletion of the tumor suppressors arid1a and pten in mice
Uploaded: 2023-02-17T13:45:00
Description: Watch this Scientific Journal Video about Non-Invasive Ultrasound Assessment of Endometrial Cancer Progression in Pax8-Directed Deletion of the Tumor Suppressors Arid1a and Pten in Mice at JoVE.com

我们感谢NCI卵巢癌孢子计划P50CA228991，博士后培训计划5T32OD011089和约翰霍普金斯大学Richard W. TeLinde捐赠基金的资助。该项目的部分资金还来自日本私立学校促进和互助公司对私立高等教育机构的经常性支出补贴。

所有涉及小鼠的程序和实验均按照约翰霍普金斯大学动物护理和使用委员会批准的方案进行。对于所有程序，都穿着适当的个人防护装备，包括手套和一次性隔离衣。处理锐器时采取了预防措施，使用后立即妥善处理在红箱锐器容器中。有关本协议中使用的所有材料和设备的详细信息，请参阅 材料表 。
1.用多西环素诱导iPAD小鼠（诱导 Pten，Arid1a 双缺?...

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A., 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=Continuing+education+course+#1:+Non-invasive+imaging+as+a+problem-solving+tool+and+translational+biomarker+strategy+in+toxicologic+pathology.">Continuing education course #1: Non-invasive imaging as a problem-solving tool and translational biomarker strategy in toxicologic pathology.</a> Toxicologic Pathology. 39 (1), 267-272 (2011).</li><li>Pfeifer, L. F., Adams, G. P., Pierson, R. 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F., 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=Ultrasound-guided+intrauterine+injection+of+lipopolysaccharide+as+a+novel+model+of+preterm+birth+in+the+mouse.">Ultrasound-guided intrauterine injection of lipopolysaccharide as a novel model of preterm birth in the mouse.</a> The American Journal of Pathology. 185 (5), 1201-1206 (2015).</li><li>Wang, T., 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=Ultrasonography+in+experimental+reproductive+investigations+on+rats.">Ultrasonography in experimental reproductive investigations on rats.</a> Journal of Visualized Experiments. 130, e56038 (2017).</li><li>Suryo Rahmanto, Y., 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=Inactivation+of+Arid1a+in+the+endometrium+is+associated+with+endometrioid+tumorigenesis+through+transcriptional+reprogramming.">Inactivation of Arid1a in the endometrium is associated with endometrioid tumorigenesis through transcriptional reprogramming.</a> Nature Communications. 11, 2717 (2020).</li><li>Pani, F., 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=Pre-existing+thyroiditis+ameliorates+papillary+thyroid+cancer:+Insights+from+a+new+mouse+model.">Pre-existing thyroiditis ameliorates papillary thyroid cancer: Insights from a new mouse model.</a> Endocrinology. 162 (10), bqab144 (2021).</li></ol>

该协议检查了超声在评估小鼠子宫腺癌进展中的子宫形态变化的效用。在这项研究中，通过纵向跟踪小鼠子宫内膜癌的诱导，发现超声检测到的解剖细节是大体和组织病理学的指标。这为使用纵向研究打开了大门，在多个时间点通过超声监测较少数量的小鼠来跟踪小鼠子宫癌的进展。这种纵向检测是通过使用手持式探头完成的，无需使用轨道系统超声设备。使用的高频探头（换能器）广泛使用，?...

小鼠仍然是生殖疾病最重要的动物模型之一1，2，3。卵巢癌和子宫癌有几种转基因或诱导的啮齿动物模型。这些研究通常依赖于在不同时间点安乐死的多个队列来捕捉形态和病理变化的纵向趋势。这阻止了在单个小鼠中获取有关癌症发展的连续数据的能力。此外，在不知道个体小鼠疾病进展状态的情况下，干预研究基于预定的时间点和先前队列的平均结果，而不是检测特定动物进展的个体阈值4，5。因此，需要允许对活体动物进行纵向评估的成像方法，以促进用于测试新药或化合物的临床前模型，并加速对病理生物学的理解，同时提高严谨性和可重复性6。
超声成像（US）是纵向监测小鼠子宫癌进展的一种有吸引力的方法，因为与其他成像方法相比，它相对简单且便宜，易于执行，并且可以具有显着的分辨率6，7。这种非侵入性方式可以在清醒小鼠或短暂镇静下的小鼠中使用5-10分钟的检查捕获微米级的特征。超声显微镜已被验证为测量小鼠卵巢卵泡发育8和植入或诱导的肿瘤生长的方法9，10，11。高频US也被用于经皮宫内注射12和观察大鼠子宫在发情周期的变化13。高频US可用于固定在专用固定平台上的小鼠，使用导轨系统固定换能器/探头，以捕获具有标准化位置和压力的高分辨率图像;但是，并非所有机构都提供此设备。手持式换能器扫描方法可以采用较少的专用设备，并用于小鼠的临床诊断和研究应用。
问题仍然是，使用手持式高频探头进行美国成像是否可以用于监测数周的子宫癌发展。与肠道类似，啮齿动物子宫是一种薄壁细长的结构，在腹部内非常移动，并且通过多个组织深度连续，这使得成像比相对不动的器官（如肾脏）更具挑战性。本研究旨在建立超声观察到的组织与组织病理学之间的相关性，确定定位小鼠子宫的标志，并确定子宫内膜癌纵向评估的可行性。这项研究提供的数据显示了US成像的子宫外观与组织病理学之间的定性对应关系，以及小鼠在几周内的连续成像。这些结果表明，手持式US可用于监测小鼠子宫内膜癌的发展，从而为收集单个小鼠纵向数据以研究子宫癌创造了机会，而无需专用设备。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagents and Equipment Used for Animal Care</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rodent Diet (2018, 625 Doxycycline)</td>
			<td>Envigio</td>
			<td>TD.01306</td>
			<td>Mouse Feed</td>
		</tr>
		<tr>
			<td>Reagents and Equipment Used for Ultrasound Imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL injectable 0.9% NaCl&#160;</td>
			<td>Hospira, Inc</td>
			<td>RL-7302</td>
			<td>Isotonic Fluid</td>
		</tr>
		<tr>
			<td>Absorbent Pad with Plastic Backing</td>
			<td>Daigger</td>
			<td>EF8313</td>
			<td>Absorbant Pads</td>
		</tr>
		<tr>
			<td>Anesthesia Induction Chambers</td>
			<td>Harvard Apparatus</td>
			<td>75-2029</td>
			<td>Induction Chamber</td>
		</tr>
		<tr>
			<td>Anesthetic absorber kit with absorber canister, holder, tubing, &#38; adapters</td>
			<td>CWE, Inc</td>
			<td>13-20000</td>
			<td>Nose Cone and Tubing</td>
		</tr>
		<tr>
			<td>Aquasonic Clear Ultrasound Gel (0.25 Liter)</td>
			<td>Parker Laboratoies</td>
			<td>08-03</td>
			<td>Ultrasound Gel</td>
		</tr>
		<tr>
			<td>BD Plastipak 3 mL Syringe</td>
			<td>BD Biosciences</td>
			<td>309657</td>
			<td>Syringe</td>
		</tr>
		<tr>
			<td>F/Air Scavenger Charcoal Canister</td>
			<td>OMNICON</td>
			<td>80120</td>
			<td>Scavenging System for Anesthesia</td>
		</tr>
		<tr>
			<td>Isoflurane, USP</td>
			<td>Vet One</td>
			<td>502017</td>
			<td>Anesthesia Agent</td>
		</tr>
		<tr>
			<td>M1050 Non-Rebreathing Mobile Anesthesia Machine</td>
			<td>Scivena Scientific</td>
			<td>M1050</td>
			<td>Anestheic Vaporizer</td>
		</tr>
		<tr>
			<td>MX550S, 25-55 MHz Transducer, 15mm, Linear</td>
			<td>VisualSonics</td>
			<td>MX550S</td>
			<td>Ultrasound Transducer (Probe)</td>
		</tr>
		<tr>
			<td>Nair Hair Aloe &#38; Lanolin Hair Removal Lotion - 9.0 oz</td>
			<td>Nair</td>
			<td></td>
			<td>Depilliating Cream</td>
		</tr>
		<tr>
			<td>Philips Norelco Multigroomer All-in-One Trimmer Series 7000</td>
			<td>Philips North America</td>
			<td>MG7750</td>
			<td>Clippers</td>
		</tr>
		<tr>
			<td>PrecisionGlide 25 G 1&#34; Needle</td>
			<td>BD Biosciences</td>
			<td>305125</td>
			<td>Needle</td>
		</tr>
		<tr>
			<td>Puralube Ophthalmic Ointment</td>
			<td>Dechra</td>
			<td>17033-211-38</td>
			<td>Lubricating Eye Drops</td>
		</tr>
		<tr>
			<td>Vevo 3100 Imaging System</td>
			<td>VisualSonics</td>
			<td>Vevo 3100</td>
			<td>Ultrasound Machine</td>
		</tr>
		<tr>
			<td>Vevo LAB 5.6.1</td>
			<td>VisualSonics</td>
			<td>Vevo LAB 5.6.1</td>
			<td>Ultrasound Analysis Software</td>
		</tr>
		<tr>
			<td>Vinyl Heating Pad with cover, 12 x 15&#34;</td>
			<td>Sunbeam</td>
			<td>731-500-000R</td>
			<td>Heating Pad</td>
		</tr>
		<tr>
			<td>Wd Elements 2TB Basic Storage</td>
			<td>Western Digital Elements</td>
			<td>WDBU6Y0020BBK-WESN</td>
			<td>Data Storage</td>
		</tr>
		<tr>
			<td>Reagents and Equipment Used for Immunohistochemistry</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10% w/v Formalin</td>
			<td>Fischer Scientific</td>
			<td>SF98-4</td>
			<td>Tissue Fixation Buffer</td>
		</tr>
		<tr>
			<td>Animal-Free Blocker and Diluent, R.T.U.</td>
			<td>Vector Laboratories Inc.</td>
			<td>&#160;SP5035</td>
			<td>Antibody Blocker</td>
		</tr>
		<tr>
			<td>Charged Super Frost Plus Glass Slides</td>
			<td>VWR</td>
			<td>4831-703</td>
			<td>Tissue Mounting Slides</td>
		</tr>
		<tr>
			<td>Citrate Buffer</td>
			<td>MilliporeSigma</td>
			<td>&#160;C9999-1000ML</td>
			<td>Epitope Retrival Buffer (pTEN)</td>
		</tr>
		<tr>
			<td>Cytoseal &#8211; 60</td>
			<td>Thermo Scientific</td>
			<td>8310-4</td>
			<td>Resin for Slide Sealing</td>
		</tr>
		<tr>
			<td>Gold Seal Cover Glass</td>
			<td>Thermo Scientific</td>
			<td>3322</td>
			<td>Coverslide</td>
		</tr>
		<tr>
			<td>Harris Modified Hematoxylin</td>
			<td>MilliporeSigma</td>
			<td>HHS32-1L</td>
			<td>Counterstain Buffer</td>
		</tr>
		<tr>
			<td>Hybridization Incubator (Dual Chamber)</td>
			<td>Fischer Scientific</td>
			<td>13-247-30Q</td>
			<td>Oven to Melt Parraffin</td>
		</tr>
		<tr>
			<td>ImmPACT DAB Substrate, Peroxidase (HRP)</td>
			<td>Vector Laboratories Inc.</td>
			<td>SK-4105</td>
			<td>Signal Development Substrate</td>
		</tr>
		<tr>
			<td>ImmPRESS HRP Goat Anti-Rabbit IgG Polymer Detection Kit, Peroxidase</td>
			<td>Vector Laboratories Inc.</td>
			<td>MP-7451</td>
			<td>Secondary IHC Antibody</td>
		</tr>
		<tr>
			<td>Oster 5712 Digital Food Steamer</td>
			<td>Oster</td>
			<td>5712</td>
			<td>Vegetable Steamer for Epitope Retrival</td>
		</tr>
		<tr>
			<td>rabbit mAB anti-ARID1a</td>
			<td>abcam</td>
			<td>ab182560</td>
			<td>Primary IHC Antibody (1:1,000)</td>
		</tr>
		<tr>
			<td>rabbit mAB anti-PTEN</td>
			<td>Cell Signaling</td>
			<td>9559</td>
			<td>Primary IHC Antibody (1:100)</td>
		</tr>
		<tr>
			<td>Scotts Tap Water Substitute</td>
			<td>MilliporeSigma</td>
			<td>S5134-100ML</td>
			<td>&#34;Blueing&#34; Buffer</td>
		</tr>
		<tr>
			<td>Tissue Path IV Cassette</td>
			<td>Fischer Scientific</td>
			<td>22272416</td>
			<td>Tissue Fixation Cassette</td>
		</tr>
		<tr>
			<td>Trilogy Buffer</td>
			<td>Cell Marque</td>
			<td>&#160;920P-10</td>
			<td>Epitope Retrival Buffer (ARID1a)</td>
		</tr>
	</tbody>
</table>

non-invasive ultrasound assessment of endometrial cancer progression in pax8-directed deletion of the tumor suppressors arid1a and pten in mice

由于这些模型中易于处理和遗传操作，可以在小鼠中研究子宫癌。然而，这些研究通常仅限于评估在不同队列的多个时间点安乐死的动物的病理学死后，这增加了研究所需的小鼠数量。纵向研究中的小鼠成像可以跟踪个体动物的疾病进展，减少所需的小鼠数量。超声技术的进步使得检测组织中的微米级变化成为可能。超声已被用于研究卵巢中的卵泡成熟和异种移植物的生长，但尚未应用于小鼠子宫的形态变化。该协议检查了诱导子宫内膜癌小鼠模型中病理学与 体内 成像比较的并置。超声观察到的特征与大体病理学和组织学观察到的变化程度一致。发现超声波对观察到的病理具有高度预测性，支持将超声检查纳入子宫疾病（如小鼠癌症）的纵向研究中。

Pax8-Cre-Arid1a-Pten 双缺失 （iPAD） 转基因小鼠维持在混合遗传背景 （129S， BALB/C， C57BL/6） 上，如前所述14.小鼠均饲喂多西环素饲料2周以诱导Cre重组酶。在我们小组以前的工作中，多西环素通过管饲法给药14;然而，在目前的研究中，多西环素饲料诱导方法有效地工作并降低了小鼠的管饲压力。重要的是要检查多西环素给药方法是否足以诱导Cre重组酶...

Watch this Scientific Journal Video about Non-Invasive Ultrasound Assessment of Endometrial Cancer Progression in Pax8-Directed Deletion of the Tumor Suppressors Arid1a and Pten in Mice at JoVE.com

Non-Invasive Ultrasound Assessment of Endometrial Cancer Progression in Pax8-Directed Deletion of the Tumor Suppressors Arid1a and Pten in Mice

该协议描述了一种使用与大体和组织学变化相关的超声成像在子宫内膜癌诱导小鼠模型中监测子宫中形态变化随时间进展的方法。

小鼠中Pax8定向缺失肿瘤抑制因子 Arid1a 和 Pten 的子宫内膜癌进展的非侵入性超声评估

Uterine cancers can be studied in mice due to the ease of handling and genetic manipulation in these models. However, these studies are often limited to ...

这项研究表明，手动引导超声非常适合监测子宫内膜癌诱导小鼠模型中子宫病理的发展。超声成像是无创的，因此可以在几天和几周内对个体小鼠进行纵向研究。这允许捕获个体变异，并最终减少用于研究的动物。该方法可以很容易地应用于监测病理变化和其他镜像模型，包括用于泌尿和消化道肿瘤形成和慢性疾病的模型。通过选择范围为 32 至 56 兆赫兹的换能器或探头，开始设置超声机以对子宫或卵巢进行成像。从主屏幕转到“应用程序”选项卡，然后选择鼠标小腹部模式。然后单击“扫描”返回主屏幕并等待显示实时图像。接下来，从左侧工具栏上的选项中选择 B 模式，然后单击“更多控件”选项以查看用于图像细化的其他工具，例如图像增益和深度或调整剪辑采集设置，例如每秒帧数。选择图像设置后，单击B模式返回主屏幕。将装有无菌等渗液体溶液的注射器放在加热垫和吸收垫之间几分钟，将其预热至35至40摄氏度。如果机器没有加热器，请在加热垫上放一瓶超声波凝胶。要将一到两毫升溶液注入小鼠的腹膜腔，请用一只手固定小鼠，露出腹侧。然后将鼠标保持在大约 20 度角，鼻子指向地板。将 25 号结核菌素注射器针头插入腹部尾部右象限的皮肤和腹壁。为避免注射到脉管系统或胃肠道中，请以最小的压力拉回注射器柱塞以寻找血液。将鼠标置于加热垫上的吸收垫上的腹侧卧位，然后将啮齿动物鼻锥牢固地放在鼠标的鼻子和口吻上。在麻醉小鼠的最后一根肋骨和骨盆之间的两侧将少量预热的超声凝胶远轴到脊柱。将少量凝胶放在超声探头上。接下来，将探针平行于椎骨放置，探针的前部位于颅侧。将鼠标放在腹侧卧位，慢慢扫描该区域以查找肾脏标志。在看到肾脏的情况下，拉动探头尾部以找到卵巢，卵巢是一个略微高回声的椭圆形到圆形结构，位于一个非常高回声的卵巢脂肪垫内，颅腹与肾脏接壤，背侧与背腹壁接壤。通过使用控制屏幕底部的滑块调整信号增益来提高图像对比度。为了改善肾脏的成像，用手指对侧腹部施加压力。改变压力和角度，从平行于脊柱到大约 20 度腹侧。查看感兴趣的器官后，通过单击捕获视频 保存剪辑 or 开始录制.完成后，单击“停止录制”以预设的帧数保留图像。将探头向尾部握住，直到卵巢位于视野的最颅面。改变探头压力和角度以查看和成像子宫。对每个感兴趣的器官重复视频和帧收集。发现子宫沿腹背壁纵向延伸，腿部外侧肌肉组织一览无余。监测组织的蠕动运动，以区分肠袢和子宫固定角。将鼠标放在背卧位以从腹侧方法收集图像。确保眼睛润滑充分，并且枪口固定在鼻锥中。在腹腹处涂抹少量预热的超声凝胶，然后在耻骨的中线和颅骨处使用探头，将膀胱定位为低回声标志。要找到子宫角，请将探头拉向膀胱的侧面。从鼠标的一侧或两侧施加轻微的数字压力。一旦子宫角进入视野，将探头垂直于鼠标并扫描腹部两侧以捕获两个角的横向视图。旋转探头以捕获矢状视图。超声波检查后，用纸巾擦拭鼠标，然后将其放回笼子中恢复。一旦小鼠在两到五分钟后完全清醒，将鼠标送回动物室。盐水注射前后小鼠腹腔的超声图像比较显示，盐水注射后腹部器官（包括子宫和卵巢）的可视化得到改善。腹侧方法用于研究与低回声膀胱相邻的子宫角的横截面。使用针对Arid1a和Pten的抗体进行免疫组织化学，以研究与邻近基质组织相比，子宫上皮细胞中完成的足够的肿瘤抑制蛋白损失。在多西环素治疗三周后，检测到具有早期病理变化的小鼠子宫的超声图像，显示子宫腔的低回声和扩张。在同一子宫的大体病理学和组织病理学研究中，分别观察到较小但扩张的薄壁子宫和保留明显管腔的早期子宫内膜增生。在较晚的时间点安乐死的小鼠显示出较大的子宫，较小的管腔和增厚的壁。在学习使用手动放置探针时，过大的压力或快速移动会使器官识别变得困难。使用温和、深思熟虑的动作，并专注于首先识别具有里程碑意义的器官。在研究终点，处死小鼠并收集组织以确认基因敲低并表征组织变化。该技术允许研究人员随着时间的推移监测腹内病变。在我们的案例中，我们用它来监测iPad模型中的子宫癌进展，然后我们通过组织病理学证实了这一点。

Research

JoVE Journal

Cancer Research

Non-Invasive Ultrasound Assessment of Endometrial Cancer Progression in Pax8-Directed Deletion of the Tumor Suppressors Arid1a and Pten in Mice

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

该<em&gt;果蝇</em&gt;成虫盘瘤模型：基因的表达与肿瘤侵袭性的可视化和定量遗传马赛克

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

科研

癌症研究

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

体内在肿瘤微环境中 pH 值、 pO2、氧化还原状态、磷酸盐和谷胱甘肽浓度的 EPR 评价

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models

核素荧光报告基因成像在啮齿动物肿瘤模型中跟踪肿瘤进展

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis ...

Utilization of Ultrasound Guided Tissue-directed Cellular Implantation for the Establishment of Biologically Relevant Metastatic Tumor Xenografts

超声引导组织定向细胞植入术在生物学相关转移性肿瘤移植中的应用

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard ...

Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors

肿瘤免疫和非免疫微环境在小鼠皮下肿瘤中的富集与鉴定

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

数字聚合酶链反应法测定零星家族性腺瘤性息肉病患者的基因变异

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

Intramucosal Inoculation of Squamous Cell Carcinoma Cells in Mice for Tumor Immune Profiling and Treatment Response Assessment

小鼠鳞状细胞细胞的肿瘤免疫分析及治疗反应评价

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop ...

In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice

在微RNA的Vivo抑制下，降低小鼠的肿瘤生长

MicroRNAs (miRNAs) are important regulators of gene expression through their ability to destabilize mRNA and inhibit translation of target mRNAs. An ...

Detection of Lung Tumor Progression in Mice by Ultrasound Imaging

超声成像对小鼠肺肿瘤进展的检测

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic ...