Name: intestinal epithelial regeneration in response to ionizing irradiation
Uploaded: 2022-07-27T14:05:00
Description: Watch this Scientific Journal Video about Intestinal Epithelial Regeneration in Response to Ionizing Irradiation at JoVE.com

作者希望感谢石溪癌症中心组织学研究核心在组织标本制备方面的专家协助，以及石溪大学实验动物资源部在动物护理和处理方面的协助。这项工作得到了美国国立卫生研究院DK124342的资助，授予Agnieszka B. Bialkowska和DK052230授予Vincent W. Yang博士。

所有小鼠都被安置在石溪大学的实验动物资源部（DLAR）。石溪大学机构动物护理和使用委员会（IACUC）批准了所有涉及动物受试者的研究和程序。涉及动物受试者的实验严格按照批准的动物处理方案（IACUC #245094）进行。
注意：商业上获得小鼠品系B6;129-Bmi1 tm1（cre/ERT）Mrc/J（Bmi1-Cre ER）和B6.129X1-Gt（ROSA）26Sortm1（EYFP）Cos/J（Rosa26eYFP）（?...

该协议描述了一个稳健且可重复的辐射损伤模型。它允许精确分析损伤后7天内肠上皮的变化。重要的是，所选的时间点反映了损伤的关键阶段，其特征是肠道的明显改变（损伤、细胞凋亡、再生和正常化阶段）60。已经建立并仔细评估了这种照射模型，证明了一种合适的损伤表现，以模仿接受放射治疗的患者所经历的损伤表现61。超过一半的结直肠癌和妇科癌?...

如果完全平放，人类的肠上皮将覆盖大约半个羽毛球场的表面1.相反，这种将人类与肠道内容物分开的单细胞层被压缩成一系列手指状的突起、绒毛和凹痕，这些隐窝使肠道的表面积最大化。上皮细胞沿隐窝绒毛轴分化。绒毛主要由吸收营养的小肠细胞、分泌粘液的高脚杯细胞和产生激素的小肠内分泌细胞组成，而隐窝主要由产生防御素的潘氏细胞、活性和储备干细胞以及祖细胞2，3，4，5组成。此外，这些细胞与底层间充质区室的基质和免疫细胞以及腔内微生物群的双向通信产生了维持肠道稳态的复杂相互作用网络，并且对损伤后的恢复至关重要6，7，8。
肠上皮是人体内自我更新最迅速的组织，周转率为2-6天9，10，11。在体内平衡期间，位于肠隐窝基部的活性干细胞（隐窝基柱状细胞）以表达富含亮氨酸的重复含 G 蛋白偶联受体 5 （LGR5） 为标志，快速分裂并提供分化为所有其他肠上皮谱系的祖细胞。然而，由于其高有丝分裂率，活性干细胞及其直系祖细胞对伽马辐射损伤特别敏感，并在照射后发生细胞凋亡5，12，13，14。在它们丢失时，肠隐窝内的储备干细胞和非干细胞（祖细胞亚群和一些终末分化细胞）经历激活并补充基底隐窝隔室，然后可以重建绒毛的细胞群，从而再生肠上皮15。使用谱系追踪技术，多个研究小组已经证明，储备（静止）干细胞能够在活性干细胞丢失时支持再生13，16，17，18，19，20，21，22。这些细胞的特征在于存在多梳复合蛋白 1 癌基因 （Bmi1）、小鼠端粒酶逆转录酶基因 （mTert）、啤酒花同源盒 （Hopx） 和富含亮氨酸的重复蛋白 1 基因 （Lrig1）。此外，已经表明非干细胞能够在受伤时补充肠道隐窝23，24，25，26，27，28，29，30，31。特别是，已经表明分泌细胞和肠细胞的祖细胞在损伤时经历去分化，恢复为干细胞样细胞，并支持肠上皮的再生。最近的研究已经确定了表达多种标记物的细胞，这些标记物具有在损伤时获得干样特征的能力（例如DLL +，ATOH1 +，PROX1 +，MIST1 +，DCLK1 +）32，33，34，35，36。令人惊讶的是，Yu等人表明，即使是成熟的潘氏细胞（LYZ+）也可以促进肠道再生37。此外，除了引起肠上皮细胞凋亡和破坏上皮屏障功能外，照射还会导致肠道菌群失调，免疫细胞活化和促炎反应的启动，以及间充质和基质细胞的活化38，39。
伽马辐射是癌症治疗中有价值的治疗工具，特别是对于结直肠肿瘤40。然而，照射通过诱导细胞损伤来显着影响肠道稳态，从而导致细胞凋亡。辐射暴露会导致多种扰动，从而减慢患者的康复速度，并以急性期粘膜损伤和炎症以及长期腹泻、尿失禁、出血和腹痛为特征。这一系列表现被称为胃肠道辐射毒性。此外，辐射诱导的透壁纤维化和/或血管硬化的进展可能仅在治疗后数年出现38，41。在损伤本身的同时，辐射在肠道细胞中诱导修复反应，激活负责启动和协调再生的信号通路42。放射诱发的小肠疾病可起源于向其他器官（如子宫颈、前列腺、胰腺、直肠）提供的盆腔或腹部放疗41，43，44，45，46。因此，肠道照射损伤是一个重要的临床问题，更好地了解由此产生的病理生理学可能会推动干预措施的发展，以减轻与放疗相关的胃肠道并发症。除了辐射之外，还有其他技术可以研究肠上皮的再生目的。已经开发了用于研究炎症和此后再生的转基因和化学小鼠模型47。葡聚糖硫酸钠（DSS）诱导肠道炎症，并导致与炎症性肠病相似的特征的发展48。DSS治疗与促癌化合物嘧啶甲烷（AOM）的组合可导致结肠炎相关癌症的发展48，49。缺血再灌注诱导损伤是另一种用于研究肠上皮再生电位的方法。这种技术需要经验和外科知识50.此外，上述技术引起的伤害类型与辐射不同，并可能导致不同再生机制的参与。此外，这些模型非常耗时，而辐射技术相当简短。最近，利用肠和结肠产生的肠和结肠样的体外方法已被与辐射损伤结合使用，以研究肠道再生的机制51，52。然而，这些技术并不能完全概括它们建模的器官53，54。
提出的方案包括对γ辐射损伤小鼠模型的描述，以及结合遗传模型，在他莫昔芬治疗后，允许追踪源自储备干细胞群的谱系（Bmi1-CreER;罗莎26eYFP）。该模型利用 12 Gy 全身照射，诱导足够显着的肠道损伤以激活储备干细胞，同时仍允许在受伤后 7 天内对肠道再生能力进行后续研究55。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL syringe</td>
			<td>BD</td>
			<td>309659</td>
			<td>-</td>
		</tr>
		<tr>
			<td>16G Reusable Small Animal Feeding Needles: Straight</td>
			<td>VWR</td>
			<td>20068-630</td>
			<td>-</td>
		</tr>
		<tr>
			<td>27G x 1/2&#34; needle</td>
			<td>BD</td>
			<td>305109</td>
			<td>-</td>
		</tr>
		<tr>
			<td>28G x 1/2&#34; Monoject 1mL insulin syringe</td>
			<td>Covidien</td>
			<td>1188128012</td>
			<td>-</td>
		</tr>
		<tr>
			<td>5-Ethynyl-2&#8242;-deoxyuridine (EdU)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc284628A</td>
			<td>10 mg/mL in sterile DMSO:water (1:4 v/v), aliquot and store in -20&#176;C</td>
		</tr>
		<tr>
			<td>Azer Scientific&#160;10% Neutral Buffered Formalin</td>
			<td>Fisher Scientific</td>
			<td>22-026-213</td>
			<td>-</td>
		</tr>
		<tr>
			<td>B6.129X1-Gt(ROSA)26Sortm1(EYFP)Cos/J</td>
			<td>The Jackson Laboratory</td>
			<td>Strain #:006148</td>
			<td></td>
		</tr>
		<tr>
			<td>B6;129-Bmi1tm1(cre/ERT)Mrc/J</td>
			<td>The Jackson Laboratory</td>
			<td>Strain #:010531</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin Fraction V, heat shock</td>
			<td>Millipore-Sigma</td>
			<td>3116956001</td>
			<td></td>
		</tr>
		<tr>
			<td>Chicken anti-GFP</td>
			<td>Aves</td>
			<td>GFP-1020</td>
			<td></td>
		</tr>
		<tr>
			<td>Click-IT plus EdU Alexa Fluor 555 imaging kit, Invitrogen</td>
			<td>Thermo Fisher Scientific</td>
			<td>C10638</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Corn oil</td>
			<td>Millipore-Sigma</td>
			<td>C8267</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Decloaking Chamber</td>
			<td>Biocare Medical</td>
			<td>DC2012</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Fisher BioReagents</td>
			<td>BP231-100</td>
			<td>light sensitive</td>
		</tr>
		<tr>
			<td>DNase-free proteinase K</td>
			<td>Invitrogen</td>
			<td>C10618H</td>
			<td>diluted 25x in DPBS</td>
		</tr>
		<tr>
			<td>Donkey anti-chicken AF647</td>
			<td>Jackson ImmunoResearch</td>
			<td>703-605-155</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Fisher Scientific</td>
			<td>21-031-CV</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Eosin</td>
			<td>Fisher Scientific</td>
			<td>S176</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence Microscope Nikon Eclipse 90i Bright and fluoerescent light, with objectives: 10X, 20X</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount Aqueous Mounting Medium</td>
			<td>Millipore-Sigma</td>
			<td>F4680-25ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Gamma Cell 40 Exactor</td>
			<td>Best Theratronics Ltd.</td>
			<td>-</td>
			<td>0.759 Gy min-1</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit AF488</td>
			<td>Jackson ImmunoResearch</td>
			<td>111-545-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin Solution, Gill No. 3</td>
			<td>Millipore-Sigma</td>
			<td>GHS332</td>
			<td></td>
		</tr>
		<tr>
			<td>HM 325 Rotary Microtome from Thermo Scientific</td>
			<td>Fisher Scientific</td>
			<td>23-900-668</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33258, Pentahydrate (bis-Benzimide)</td>
			<td>Thermo Fisher Scientific</td>
			<td>H3569</td>
			<td>dilution 1:1000</td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide Solution, ACS, 29-32%, Spectrum Chemical</td>
			<td>Fisher Scientific</td>
			<td>18-603-252</td>
			<td>-</td>
		</tr>
		<tr>
			<td>In Situ Cell Death Detection Kit, Fluorescein (Roche)</td>
			<td>Millipore-Sigma</td>
			<td>11684795910</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid Blocker Super PAP PEN, Mini</td>
			<td>Fisher Scientific</td>
			<td>DAI-PAP-S-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Lithium Carbonate (Powder/Certified ACS), Fisher Chemical</td>
			<td>Fisher Scientific</td>
			<td>L119-500</td>
			<td>0.5g/1L dH2O</td>
		</tr>
		<tr>
			<td>Luer-Lok Syringe sterile, single use, 10 mL</td>
			<td>VWR</td>
			<td>89215-218</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>VWR</td>
			<td>BDH1135-4LP</td>
			<td></td>
		</tr>
		<tr>
			<td>Pharmco Products&#160;Ethyl alcohol, 200 PROOF</td>
			<td>Fisher Scientific</td>
			<td>NC1675398</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Pharmco-Aaper 281000ACSCSLT Acetic Acid ACS Grade</td>
			<td>Capitol Scientific</td>
			<td>AAP-281000ACSCSLT</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Rabbit anti-Ki67</td>
			<td>BioCare Medical</td>
			<td>CRM325</td>
			<td></td>
		</tr>
		<tr>
			<td>Richard-Allan Scientific Cytoseal XYL Mounting Medium</td>
			<td>Fisher Scientific</td>
			<td>22-050-262</td>
			<td></td>
		</tr>
		<tr>
			<td>Scientific Industries Incubator-Genie for baking slides at 65 degree</td>
			<td>Fisher Scientific</td>
			<td>50-728-103</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Citrate Dihydrate</td>
			<td>Fisher Scientific</td>
			<td>S279-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless Steel Dissecting Kit</td>
			<td>VWR</td>
			<td>25640-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Plus micro slides [size: 25 x 75 x 1 mm]</td>
			<td>VWR</td>
			<td>&#160;48311-703</td>
			<td></td>
		</tr>
		<tr>
			<td>Tamoxifen</td>
			<td>Millipore-Sigma</td>
			<td>T5648</td>
			<td>30 mg/mL in sterile corn oil, preferably fresh or short-sterm storage in -20&#176;C, light sensitive</td>
		</tr>
		<tr>
			<td>Tissue-Tek 24-Slide Holders with Detachable Handle</td>
			<td>Sakura</td>
			<td>4465</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek&#160;Accu-Edge&#160;Low Profile Blades</td>
			<td>Sakura</td>
			<td>4689</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek&#160;Manual Slide Staining Set</td>
			<td>Sakura</td>
			<td>4451</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek Staining Dish, Green with Lid</td>
			<td>Sakura</td>
			<td>4456</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek Staining Dish, White with Lid</td>
			<td>Sakura</td>
			<td>4457</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Millipore-Sigma</td>
			<td>P7949</td>
			<td></td>
		</tr>
		<tr>
			<td>Unisette Processing Cassettes</td>
			<td>VWR</td>
			<td>87002-292</td>
			<td>-</td>
		</tr>
		<tr>
			<td>VWR Micro Cover Glasses</td>
			<td>VWR</td>
			<td>48393-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Fisher Scientific</td>
			<td>X5P-1GAL</td>
			<td></td>
		</tr>
	</tbody>
</table>

intestinal epithelial regeneration in response to ionizing irradiation

肠上皮由单层细胞组成，但包含多种类型的终末分化细胞，这些细胞是由位于肠隐窝底部的肠道干细胞的活跃增殖产生的。然而，在急性肠损伤事件期间，这些活跃的肠道干细胞会经历细胞死亡。伽马射线照射是一种广泛使用的结直肠癌治疗方法，虽然治疗有效，但具有耗尽活性干细胞库的副作用。事实上，患者在接受放疗时经常出现胃肠道辐射综合征，部分原因是活跃的干细胞耗竭。肠隐窝中活性肠道干细胞的丢失激活了通常静止储备的肠道干细胞库，并诱导分泌细胞和肠细胞前体细胞的去分化。如果没有这些细胞，肠上皮将缺乏从放疗和其他此类主要组织损伤中恢复的能力。谱系追踪技术的新进展允许跟踪再生过程中细胞的活化、分化和迁移，并已成功用于肠道研究。本研究旨在描述一种分析辐射损伤后小鼠肠上皮内细胞的方法。

将 12 Gy 全身照射 （TBI） 与小鼠遗传谱系追踪相结合，可以对肠道辐射损伤的后果进行彻底分析。首先， Bmi1-CreER;Rosa26eYFP小鼠接受单次他莫昔芬注射，该注射液在Bmi1 + 储备干细胞群中诱导增强的黄色荧光蛋白（EYFP）表达。注射他莫昔芬两天后，小鼠接受照射或假照射。安乐死前三小时，小鼠被注射EdU。安乐死后，收集小肠标本在照射后0 h、3 h、6 h、24 h、48 h、72 h?...

Watch this Scientific Journal Video about Intestinal Epithelial Regeneration in Response to Ionizing Irradiation at JoVE.com

Intestinal Epithelial Regeneration in Response to Ionizing Irradiation

胃肠道是放射治疗癌症治疗后对损伤最敏感的器官之一。它同时也是一个器官系统，在这种侮辱之后具有最高的再生能力。所提出的协议描述了一种研究肠上皮再生能力的有效方法。

响应电离照射的肠上皮再生

The intestinal epithelium consists of a single layer of cells yet contains multiple types of terminally differentiated cells, which are generated by the ...

伽马射线照射是一种广泛使用的结直肠癌治疗方法。虽然它在治疗上有效，但它对胃肠道有负面影响。所提出的协议描述了一种有效的方法来研究辐射损伤后再生过程中细胞的活化，分化和迁移。该协议描述了一个稳健且可重复的辐射损伤模型。重要的是，该模型允许跟踪损伤后的肠道细胞命运，从而更好地了解特定细胞亚群的再生能力。该技术可以采用不同的谱系追踪动物模型来研究损伤后细胞各种亚群的实时行为和相互作用。对该方案的微小修改应允许研究辐射损伤时微生物群与不同上皮、基质和免疫细胞群之间的关系。首先，将他莫昔芬粉末重悬于无菌玉米油中，浓度为每微升30微克。在 30 秒的开关周期内以 60% 的振幅进行超声处理三分钟。在室温下在黑暗中旋转混合一小时。然后，通过将无菌DMSO添加到EdU粉末中来制备EdU。加入总体积的1/5，慢慢加入剩余体积的无菌超纯水。完全溶解后，等分并储存在20摄氏度。准备用于组织收集和固定的试剂，例如 70% 乙醇水溶液、冷却至 4 摄氏度的 DPBS、Bouin 固定缓冲液和 10% 缓冲福尔马林。接下来，准备必要的设备，例如小鼠解剖试剂盒，培养皿，连接到10毫升注射器的16号管饲针和组织学盒。在伽马辐照暴露前两天，准备实验动物注射他莫昔芬。称量每只动物，并计算所需剂量。用70%乙醇消毒腹部区域。然后，使用连接到 1 毫升注射器的 27 号针头腹膜内注射单剂量他莫昔芬，以诱导 Cre 介导的重组。在接下来的48小时内观察动物以排除潜在的他莫昔芬毒性。48小时后，将动物转移到照射室。用70%乙醇溶液对伽马辐照器中的样品室进行消毒。将吸收垫和动物放在样品室内。盖上盖子，然后关闭腔室。对伽马辐照器进行编程，使动物暴露于 12 个灰色全身照射下。然后，将钥匙转到起始位置，然后按开始开始曝光。离开房间进行主动曝光时间。经过预设时间后，机器将自动停止并开始发出哔哔声。通过将钥匙转到停止位置来关闭机器。打开样品室，取下盖子。将动物转移到笼子里，并用70%乙醇对样品室进行消毒。将动物转移回传统的住房房间，并在治疗后观察它们的状况。每天监测动物的体重。在计划的安乐死前三小时，对腹部区域进行消毒，并使用28号胰岛素注射器腹膜内注射100微升EdU储备溶液。对动物实施安乐死后，在照射后的不同时间点收集近端肠。解剖小肠的近端，并去除附着的组织。使用连接到 10 毫升注射器的 16 号直进纸针用冷 DPBS 冲洗组织，并用 Bouin 的固定缓冲液固定组织。纵向切开肠子，然后使用瑞士卷技术滚动近端肠。将组织置于组织学盒中，置于装有10%缓冲福尔马林的容器中，并在室温下孵育24至48小时。孵育后，将组织学盒转移到70%乙醇中，并将组织嵌入石蜡中。包埋后，将组织学盒放在冰上至少一小时。然后，使用切片机切割五微米厚的水平切片。将组织切片转移到加热至45摄氏度的水浴中，并将切片放在带电载玻片上。将载玻片放在架子上在室温下过夜晾干。小肠标本的苏木精和伊红图像显示0小时时肠上皮稳态，随后在凋亡期隐窝隔室内细胞丢失。随后是再生阶段，其中高度增殖的细胞填充了隐窝，导致第七天进入正常化阶段。EYFP的谱系追踪表明，在体内平衡期间，来自Bmi1阳性储备干细胞的细胞被限制在隐窝内的正四到六位。EdU和Ki-67染色显示假照射小鼠的肠道稳态正常，从活性干细胞延伸到转运扩增区。在凋亡期，EYFP阳性细胞的数量减少，伴随着辐射损伤引起的增殖细胞的丧失。在再生阶段，观察到储备干细胞的活化和Bmi1阳性细胞的快速增殖。进一步增殖和迁移恢复了肠上皮的完整性。TUNEL 染色显示，在稳态期间不存在凋亡细胞。在凋亡期后期观察到TUNEL染色增加。在再生阶段，再生隐窝内的TUNEL染色稳步下降，并且在隐窝归一化时几乎不存在。该技术可以与RNA测序，单细胞RNA测序，荧光激活细胞分选或蛋白质组学分析相结合，以收集有关特定细胞谱系在损伤后肠道再生中的作用的更深入的知识。研究人员可以研究肠道再生中微生物群与上皮、基质和免疫细胞群之间的相互作用，以扩大对肠上皮再生能力的理解。

intestinal-epithelial-regeneration-in-response-to-ionizing-irradiation

Research

JoVE Journal

Biology

Cortical Neurogenesis: Transitioning from Advances in the Laboratory to Cell-Based Therapies

皮层神经再生：从实验室中的研究进展过渡到细胞疗法

科研

生物学

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &amp;#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

电离辐射的反应定量的γH2AX灶

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Evaluation of the Spatial Distribution of &gamma;H2AX following Ionizing Radiation

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3. The phosphorylated form of H2AX, referred to as &gamma;H2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3. Analysis and quantitation of &gamma;H2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds4.
Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of &gamma;H2AX as molecular marker of DSBs, a disparity in &gamma;-irradiation-induced &gamma;H2AX foci formation in euchromatin and heterochromatin has been observed5-7. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of &gamma;H2AX following ionizing radiation8. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between &gamma;H2AX formation and active transcription9. Here we demonstrate our immunofluorescence method for detection and quantitation of &gamma;H2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3D-modeling.

Evaluation of the Spatial Distribution of &amp;#947;H2AX following Ionizing Radiation

Microscopic analysis of &gamma;H2AX foci, which form following the phosphorylation of H2AX at Ser-139 in response to DNA double-strand breaks, has become an invaluable tool in radiation biology. Here we used an antibody to mono-methylated histone H3 at lysine 4 as an epigenetic marker of actively transcribing euchromatin, to evaluate the spatial distribution of radiation-induced &gamma;H2AX formation within the nucleus.

评价以下电离辐射的空间分布的γH2AX

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone ...

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

一个系统为培养虹膜色素上皮细胞，研究蝾螈镜头再生

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in renal failure1. AKI can be caused by a number of factors including ischemia, drug-based toxicity, or obstructive injury1. This results in an inability to maintain fluid and electrolyte homeostasis. While AKI has been observed for decades, effective clinical therapies have yet to be developed. Intriguingly, some patients with AKI recover renal functions over time, a mysterious phenomenon that has been only rudimentally characterized1,2. Research using mammalian models of AKI has shown that ischemic or nephrotoxin-injured kidneys experience epithelial cell death in nephron tubules1,2, the functional units of the kidney that are made up of a series of specialized regions (segments) of epithelial cell types3. Within nephrons, epithelial cell death is highest in proximal tubule cells. There is evidence that suggests cell destruction is followed by dedifferentiation, proliferation, and migration of surrounding epithelial cells, which can regenerate the nephron entirely1,2. However, there are many unanswered questions about the mechanisms of renal epithelial regeneration, ranging from the signals that modulate these events to reasons for the wide variation of abilities among humans to regenerate injured kidneys.
The larval zebrafish provides an excellent model to study kidney epithelial regeneration as its pronephric kidney is comprised of nephrons that are conserved with higher vertebrates including mammals4,5. The nephrons of zebrafish larvae can be visualized with fluorescence techniques because of the relative transparency of the young zebrafish6. This provides a unique opportunity to image cell and molecular changes in real-time, in contrast to mammalian models where nephrons are inaccessible because the kidneys are structurally complex systems internalized within the animal. Recent studies have employed the aminoglycoside gentamicin as a toxic causative agent for study of AKI and subsequent renal failure: gentamicin and other antibiotics have been shown to cause AKI in humans, and researchers have formulated methods to use this agent to trigger kidney damage in zebrafish7,8. However, the effects of aminoglycoside toxicity in zebrafish larvae are catastrophic and lethal, which presents a difficulty when studying epithelial regeneration and function over time. Our method presents the use of targeted cell ablation as a novel tool for the study of epithelial injury in zebrafish. Laser ablation gives researchers the ability to induce cell death in a limited population of cells. Varying areas of cells can be targeted based on morphological location, function, or even expression of a particular cellular phenotype. Thus, laser ablation will increase the specificity of what researchers can study, and can be a powerful new approach to shed light on the mechanisms of renal epithelial regeneration. This protocol can be broadly applied to target cell populations in other organs in the zebrafish embryo to study injury and regeneration in any number of contexts of interest.

Acute kidney injury (AKI) in humans is a common clinical problem caused by damage to the epithelial cells that comprise kidney nephrons, and AKI is associated with high mortality rates of 50-70%1. Following epithelial cell destruction, nephrons have a limited ability to regenerate, though the mechanisms and limitations that guide this phenomenon remain poorly understood. In this video article, we describe our technique for targeted laser ablation of kidney nephron cells in the zebrafish embryo kidney, or pronephros. Our new method can be used to complement nephrotoxicity-induced models of AKI and gain a high-resolution understanding of the cell and molecular alterations that are associated with epithelial regeneration in the kidney nephron.

激光烧蚀斑马鱼Pronephros研究肾小管上皮再生

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

RNA纯化的染色质隔离（CHIRP）

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

在毛发再生的上皮 - 间充质信号的快速遗传分析

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Measuring Glutathione-induced Feeding Response in Hydra

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. The movement of prey organisms causes mechanosensory discharge of the stinging cells called nematocysts from hydra, which are inserted into the prey. The feeding response in hydra, which includes curling of the tentacles to bring the prey towards the mouth, opening of the mouth and consequent engulfing of the prey, is triggered by GSH present in the fluid released from the injured prey. To be able to identify the molecular mechanism of the feeding response in hydra which is unknown to date, it is necessary to establish an assay to measure the feeding response. Here, we describe a simple method for the quantitation of the feeding response in which the distance between the apical end of the tentacle and mouth of hydra is measured and the ratio of such distance before and after the addition of GSH is determined. The ratio, called the relative tentacle spread, was found to give a measure of the feeding response. This assay was validated using a starvation model in which starved hydra show an enhanced feeding response in comparison with daily fed hydra.

Here we describe a simple assay for the quantification of the feeding response in hydra induced by the reduced form of glutathione. This assay relies on measuring the distance between the apical end of the tentacle and mouth of hydra.

测量水润谷胱甘肽诱导饲养响应

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. ...

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

Two methods are presented here to determine intestinal barrier function. An epithelial meter (volt/ohm) is used for measurements of transepithelial electrical resistance of cultured epithelia directly in tissue culture wells. In mice, the FITC-dextran gavage method is used to determine the intestinal permeability in vivo.

肠上皮细胞通透性的体外及体内测定方法

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and ...

Dosimetry for Cell Irradiation using Orthovoltage (40-300 kV) X-Ray Facilities

The importance of dosimetry protocols and standards for radiobiological studies is self-evident. Several protocols have been proposed for dose determination using low energy X-ray facilities, but depending on the irradiation configurations, samples, materials or beam quality, it is sometimes difficult to know which protocol is the most appropriate to employ. We, therefore, propose a dosimetry protocol for cell irradiations using low energy X-ray facility. The aim of this method is to perform the dose estimation at the level of the cell monolayer to make it as close as possible to real cell irradiation conditions. The different steps of the protocol are as follows: determination of the irradiation parameters (high voltage, intensity, cell container etc.), determination of the beam quality index (high voltage-half value layer couple), dose rate measurement with ionization chamber calibrated in air kerma conditions, quantification of the attenuation and scattering of the cell culture medium with EBT3 radiochromic films, and determination of the dose rate at the cellular level. This methodology must be performed for each new cell irradiation configuration as the modification of only one parameter can strongly impact the real dose deposition at the level of the cell monolayer, particularly involving low energy X-rays.

Dosimetry for Cell Irradiation using Orthovoltage 40-300 kV X-Ray Facilities

This document describes a new dosimetry protocol for cell irradiations using low energy X-ray equipment. Measurements are performed in conditions simulating real cell irradiation conditions as much as possible.