作者感谢阿尔康为这些研究提供财政支持。

1. 人类角膜上皮细胞培养
<ol><li>生长不朽的HCEC19在胶原蛋白涂层75厘米2烧瓶与20毫升杜尔贝科的改良鹰介质/营养混合物F-12（DMEM/F12）含有10%胎儿牛血清（FBS）和1%青霉素/链霉素在37°C与5%CO2。 注:不需要摇晃。</li>
	<li>每 2 −3 天更换一次媒体。</li>
</ol>2. 准备细胞进行测试
<ol><li>细胞几乎汇流后去除培养介质。</li>
	<li>在每个 75 厘米2 烧瓶中加入 4−6 mL 的细胞分离溶液。在37°C孵化细胞，直到细胞分离（约20−30分钟），定期在显微镜下检查。</li>
	<li>在每个 75厘米 2 烧瓶中加入 2−6 毫升的 DMEM/F12，其中包含 10% FBS。</li>
	<li>将烧瓶中的内容转移到 50 mL 离心机管中。</li>
	<li>以 450 −500 x g 的速度将细胞离心5分钟。将超自然物移出，并在预热介质中重新加热细胞。</li>
	<li>使用血细胞计数数细胞。计算包含共 1 x 105 个单元格的介质体积。</li>
	<li>在 48 口井胶原蛋白-1 涂层培养板的每口井中加入 1 x 10 5 个细胞。在井中添加足够的介质，使井中的最终媒体量为 0.5 mL 文化 DMEM/F12，FBS 为 10%。 注:人工撕裂配方可能导致细胞毒性，可以通过HCEC代谢活性的减少来测量。数据的一致性取决于在播种时向每口油井添加相同数量的 HCEC。建议48个井板的文化浓度为1×105。由于哺乳动物细胞在悬念后可以定居在离心管中，因此建议在用细胞播种板时经常通过搅拌来补充细胞，使细胞悬浮液的HCEC均匀分布。</li>
	<li>在 37 °C 和 5% CO2 中孵化细胞 24 小时。</li>
</ol>3. 无干燥协议
<ol><li>控制程序<ol>
<li>24 h 孵化后，将培养介质从盘子中删除。</li>
		<li>立即用 150 μL 的测试配方和介质控制解决方案进行治疗。</li>
		<li>在 37 °C 和 5% CO2 中孵化细胞 30 分钟。</li>
		<li>从细胞中取出测试解决方案。</li>
		<li>添加 0.5 mL 的 10% 代谢染料溶液，以测试代谢活性。在37°C和5%CO2下再孵化细胞4小时。</li>
		<li>4 h 潜伏期后，从每口井中取出 100 μL 的代谢染料溶液，并将每口 100 μL 放入 96 井板的井中。</li>
		<li>使用荧光板读卡器（材料表）测量每口油井的荧光度。将激发波长设置为 540 nm，将发射波长设置为 590 nm。</li>
	</ol>
</li>
	<li>无干燥协议（恢复程序）<ol>
<li>重复步骤 3.1.1−3.1.4。</li>
		<li>向每口井添加 0.5 毫升 DMEM/F12 介质。</li>
		<li>在 37 °C 和 5% CO2中孵化一组 18 h 的培养物。</li>
		<li>移除介质并添加 0.5 mL 的 10% 代谢染料溶液。重复步骤 3.1.6 和 3.1.7。</li>
	</ol>
</li>
</ol>4. 干燥协议
<ol><li>控制程序<ol>
<li>24 h 孵化后（步骤 2.8），从板中删除培养介质。</li>
		<li>立即用 150 μL 的测试配方和介质控制解决方案进行治疗。</li>
		<li>在 37 °C 和 5% CO2 中孵化细胞 30 分钟。</li>
		<li>从细胞中取出测试解决方案。</li>
		<li>将细胞放置在37°C和45%RH室中5分钟，以干燥细胞。</li>
		<li>添加 0.5 mL 的 10% 代谢染料溶液，以测试代谢活性。在37°C和5%CO2下再孵化细胞4小时。</li>
		<li>4 h 潜伏期后，从每口井中取出 100 μL 的代谢染料溶液，并将每口 100 μL 放入 96 井板的井中。</li>
		<li>使用荧光板读卡器测量每口油井的荧光度。将激发波长设置为 540 nm，将发射波长设置为 590 nm。</li>
	</ol>
</li>
	<li>恢复程序<ol>
<li>重复步骤 4.1.1-4.1.5。</li>
		<li>向每口井添加 0.5 毫升 DMEM/F12 介质。</li>
		<li>在 37 °C 和 5% CO2中孵化一组 18 h 的培养物。</li>
		<li>移除介质并添加 0.5 mL 的 10% 代谢染料溶液。重复步骤 4.1.7 和 4.1.8。</li>
	</ol>
</li>
</ol>5. 数据分析
<ol><li>计算媒体控制的平均荧光。使用以下公式计算样本的可行性百分比: 样品的存活率 = 测试样品荧光/介质控制荧光 x 100 注:媒体控制平均值为 100% 可行性。代谢染料本身有少量的荧光与它相关。代谢染料试剂的荧光可以在计算测试样品的活性之前从控制和测试样品荧光中减去。</li>
	<li>执行常态测试和对控制和测试样本的等值方差的测试。</li>
	<li>如果正常性测试通过，且方差相等，则执行单向 ANOVA。要确定特定组之间的显著差异，在临时测试后进行对比（即 Tukey 或 Dunnett 的多重比较测试）。</li>
	<li>如果正常性测试通过，方差不同，则执行韦尔奇的 ANOVA。要识别特定组之间的显著差异，在临时测试后进行对比（即 Dunnett 或 Games-Howell 多次比较测试）。</li>
	<li>如果正常性测试失败，请使用非参数测试（即克鲁斯卡尔-瓦利斯测试）。要识别特定组之间的显著差异，在特设测试后进行对比（即 Dunn 的测试）。</li>
</ol>

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M., Tong, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relevance+of+Lipid-Based+Products+in+the+Management+of+Dry+Eye+Disease.">Relevance of Lipid-Based Products in the Management of Dry Eye Disease.</a> Journal of Ocular Pharmacology and Therapeutics. 33 (9), 647-661 (2017).</li><li>Lievens, C., 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=Evaluation+of+an+enhanced+viscosity+artificial+tear+for+moderate+to+severe+dry+eye+disease:+A+multicenter,+double-masked,+randomized+30-day+study.">Evaluation of an enhanced viscosity artificial tear for moderate to severe dry eye disease: A multicenter, double-masked, randomized 30-day study.</a> Contact Lens &amp; Anterior Eye. 42 (4), 443-449 (2019).</li></ol>

三位作者（雷卡·兰加拉詹、霍华德·凯特尔森、拉克什曼·苏巴拉曼）是阿尔康的员工。阿尔康是该项目的唯一资金来源和赞助者。在过去的3年里，Lyndon W. Jones获得了以下公司的研究支持或讲座荣誉:阿尔康、阿勒甘、康塔马克、库珀维森、GL Chemtec、Inflamax研究、J&J视觉、梅尼康、自然之路、诺华、PS治疗、桑滕、夏尔、视觉玻璃和视觉。林登·琼斯也是阿尔康、库珀维森、J&J视觉、诺华和奥普特克的顾问和/或顾问委员会成员。以下作者名单无可披露:理查德·多、大卫·麦肯纳、阿德琳·苏科、达里尔·恩斯通、贾亚·丹塔姆。

该协议旨在确定人工撕裂溶液对细胞干燥的保护作用。最初，细胞暴露在干眼药水中，以涂覆细胞。接下来，细胞干燥，代谢活动被监测，以评估配方是否可以减轻干燥应激的有害影响。这些步骤对于了解干眼配方对角膜上皮细胞的整体生存能力的总体影响至关重要。
在从培养井中取出大部分干眼配方后，与细胞接触的剩余分子的化学成分将影响细胞干燥。有些产品含有微乳油，最大限度地减少细胞蒸发20。一项在冲洗后评估摩擦分析的研究表明，一些干眼配方的摩擦力较低，这可能是干眼配方21增强居住时间的指标。此测定的局限性之一是，虽然与其他眼药水配方相比，可以评估干燥保护，但由于评估的干燥时间短，无法确定此保护的持续时间。在体外测定中，较长的干燥时间可能需要使用比细胞单层具有整体水分含量的多层培养物。
如果温度和湿度值发生变化，可能需要调整细胞上配方的特定孵化时间和干燥时间的持续时间。选择温度，RH和气流模拟环境压力条件，可以促进干眼可能很困难，因为季节性和局部环境条件是相当可变的21。现代人类研究的环境室可以改变温度在5°C和35°C之间，RH在5%到95%22之间。使用蒸发计和紧密安装的护目镜，确定在 RH 40−45% 角膜蒸发率为 0.037 μL/cm2/min，在 RH 为 20−25%， 蒸发率为 0.065 μL/cm2/min23。如果体外模型在极低的RH条件下（如飞机15、沙漠或干燥的室内环境16）中观察配方的保护作用，可能需要缩短孵化时间，以适应细胞的增强蒸发率。在人类泪液中，存在各种来自肌瘤和其他腺体的脂质，保护眼泪免受蒸发。这些脂质有不同的熔点24。温度变化可能会影响泪液的流动性，因此在环境温度下进行的测试的蒸发率可能与在 37 °C 下进行的测试显著不同。 由于眼表面的平均温度范围为 32.9 至 36 °C25，建议在此温度范围附近进行测试。
除了本协议中使用的代谢染料（阿拉马蓝）外，还有其他化学试剂可用于评估产品配方对细胞代谢活动的影响。四环盐（MTT、XTT、MTS、WST）是可使用的替代化学品。四环盐的区别在于MTT是一个带正电荷的分子，所以它可以进入活细胞的细胞质26。MTT 在减少 NAD（P）H27后变得不溶。在阅读读盘器上的吸收性之前，必须进行溶解。其他四环盐的负电荷为28。它们必须被活细胞外的次生分子减少，因为它们不能进入细胞，不能直接与细胞内分子29相互作用。对于 XTT、MTS 和 WST-1 检测，添加电子耦合剂 1-甲氧苯丙胺甲硫酸酯 （PMS） 可提高检测30的性能。与使用四环盐的代谢检测相比，阿拉马蓝不需要额外的溶解剂或电子耦合剂。此外，与XTT、MTS或WST-1不同，阿拉马蓝穿透细胞膜，可直接通过细胞酶27、29减少。将阿拉马蓝与四色盐直接比较表明，阿拉马蓝对评估包括HCEC3、27、31、32在内的各种细胞系的代谢活动更为敏感。
其他生化终点也可以考虑通过干燥来评估人类角膜上皮细胞的保护。荧光染料可用于检测细胞膜渗透性和细胞酯酶活性1。此外，通过在细胞表面染色凋亡标记或测量细胞酶活性1，33来检测凋亡。其他检测已经确定眼科产品对HCEC紧密路口34的影响。这些检测可以利用本文中描述的干燥程序纳入未来的评估。
眼睛干涸的原因有很多。干眼可由眼泪分泌减少、眼部表面炎症增加或眼部脱水增加引起。对于蒸发性干眼使用干眼滴，防止其细胞在干燥条件下干燥的个人，可以减轻干眼的症状。一些干眼配方中的问题之一是存在可能损害眼部表面的防腐剂。伤害细胞对干眼病人没有帮助，因为它会导致更多的细胞死亡，因为受伤的细胞更容易干燥应激35。
Garrigue等人最近发表的一篇评论文章对目前 基于脂质的产品如何作用于缓解蒸发性干眼的知识进行了评估。在脂质不足的眼泪中加入脂质可以防止蒸发，从而保护细胞免受干燥。此外，腐殖质是吸引和保留水在眼表37的分子。溶液中的甘油#1和溶液中的丙二醇#3都是腐殖质。溶液#3显示了HCEC更大的保护作用，这可能是由于配方成分的脂质和腐殖质特性。
此协议旨在评估接触干眼配方和干燥应激后HCEC的代谢活性。通过使用本文中描述的方法，可以为蒸发性干眼患者开发新的非刺激性和保护性眼药水。

需要多剂量眼科解决方案中的许多不同成分来保持稳定性、pH、渗透性和有效性。然而，一些化学物质，如防腐剂或表面活性剂在眼溶液中可能会造成损害角膜上皮1，2。可使用人类角膜上皮细胞 （HCEC） 进行细胞生存能力测试，以评估这些成分在眼科配方中的潜在损害。体外检测，是特别有用的评估细胞损伤之一是阿拉马蓝检测3。在本测定中，代谢染料（即阿拉马蓝）含有雷萨祖林，作为细胞生存能力指标。在细胞呼吸过程中，通过接受电子4，雷萨祖林被简化为再苏鲁芬。再糖精的减少导致从非荧光分子转变为荧光分子，可以使用光谱荧光计5检测。
此调查使用两种不同的角膜上皮细胞治疗方法，以确定干眼产品在体外模型中的表现。在第一次评估中，细胞用这些产品治疗30分钟。治疗后，细胞在接触干眼产品后和恢复时间间隔后立即评估代谢活性。此评估确定这些产品在湿度室干燥前对细胞的影响。这些溶液中的溶液防腐剂、表面活性剂或缓冲剂可能对使用代谢染料检测到的细胞产生一些细胞毒性作用。
第二个评估确定暴露在干眼产物和干燥后细胞的代谢活性。在这项评估中，如果产品处理为细胞提供防干燥损伤的保护，细胞的代谢活性将得到维持。如果干眼产品可以涂上脂质的细胞，或者产品可能吸收周围增加水分的液体，则可能会对干燥的有害影响进行细胞保护。
该协议旨在模拟细胞环境压力的严重状况。其他研究也使用各种模型来制造这种外部压力。一些研究已经干燥了层流罩6，7，8的细胞，而其他研究已经使用了室温和各种相对湿度（RH）值9，10，11。模拟干眼状况的方法是增加孵化溶液12，13的渗透性。另一种模拟不良环境条件的体外方法是向细胞介质中加入细胞因子，以模拟干眼患者的泪液。虽然这些测试是有趣的模型，可以评估化学物质如何减少渗透应激或刺激免疫系统，但这些模型并没有直接显示化学配方如何保护细胞免受干燥压力。
干燥模型使用温度/湿度室 37 °C 和 45% RH。它模拟可能导致眼部表面蒸发的环境条件。也可以使用这种方法模拟其他极端环境条件，例如15 号机舱或16 号冬季室内环境中的低湿度。
细胞培养建模用于模拟干燥应激对人体角膜的影响。为了检测细胞应力，进行细胞生存能力测试，以确定对细胞健康的影响。可以在细胞上进行几种不同的生理测试，以确定它们是否受到压力。本测试过程中进行的分析使用市售的代谢染料（材料表）。它比许多其他细胞应力测试有优势，因为它是无毒的，可溶于生长介质，可以评估细胞没有细胞固定18。在此过程中，每次治疗后立即对细胞进行代谢活性测试，第二组细胞被放回生长介质，并在 18 小时后恢复后进行评估。恢复期后立即进行测试的原因是确定对代谢活动和导致延迟效应的细胞损伤的直接影响。此外，它提供了一个机会来评估这些配方对短期与长期损害的影响，以及配方从最初的侮辱中恢复过来/不恢复的能力。
本调查用于比较各种含脂干眼产品对HCEC代谢活动的影响，无论有没有干燥。所测试的干眼产品的成分在 材料表中描述。溶液中的成分#1是碳化甲基纤维素钠（0.5%）、甘油（1%）和多糖酸盐80（0.5%），溶液中#2它们是轻矿物油（1.0%）和矿物油（4.5%），并在溶液中#3脂质是矿物油和丙二醇是脱硫。这些脂质是配方成分，有望为HCEC提供防干燥保护。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>75 cm2 Vented Flask</td>
			<td>Corning</td>
			<td>354485</td>
			<td>This is the BioCoat brand collagen coated</td>
		</tr>
		<tr>
			<td>96 well plate</td>
			<td>costar</td>
			<td>3370</td>
			<td></td>
		</tr>
		<tr>
			<td>alamarBlue</td>
			<td>Fisher Scientific</td>
			<td>dal 1025</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 48 Well plates</td>
			<td>Corning</td>
			<td>354505</td>
			<td>This is the BioCoat brand collagen coated</td>
		</tr>
		<tr>
			<td>Cytation 5</td>
			<td>BioTek</td>
			<td>CYT5MPV</td>
			<td>Can read fluorescence from 280 - 700 nm (for assay 540/590)</td>
		</tr>
		<tr>
			<td>DMEM/F12 with L-glutamine and 15 mM HEPES</td>
			<td>Gibco</td>
			<td>11039-021</td>
			<td>There is No Phenol Red in this media</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Hycone</td>
			<td>SH30396.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Corneal Epithelial Cells</td>
			<td>University of Ottawa</td>
			<td>N/A</td>
			<td>SV40-immortalized human corneal epithelial cells from Dr. M. Griffith (Ottawa Eye Research Institute, Ottawa, Canada) and have been characterized Griffith M, Osborne R, Munger R, Xiong X, Doillon CJ, Laycock NL, Hakim M, Song Y, Watsky MA. Functional human corneal equivalents constructed from cell lines. Science. 1999;286:2169&#8211;72.</td>
		</tr>
		<tr>
			<td>Humidity/Temperature Chamber</td>
			<td>Associated Environmental Systems</td>
			<td>LH-1.5</td>
			<td>Economy Line Humidity Chamber</td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>100X concentration so add 1ml to each 99ml of media</td>
		</tr>
		<tr>
			<td>Refresh Optive Advanced (solution #1)</td>
			<td>Allergan, Inc</td>
			<td></td>
			<td>Carboxymethylcellulose Sodium (0.5%),Glycerin (1%),polysorbate 80 (0.5%),-Lubricants- Inactive ingredients-boric acid; carbomer copolymer type A; castor oil; erythritol; levocarnitine; purified water; Purite (stablized oxychloro complex)and may contain sodium hydroxide to adjust pH.</td>
		</tr>
		<tr>
			<td>Soothe XP (solution #2)</td>
			<td>Bausch &#38; Lomb</td>
			<td></td>
			<td>Light mineral oil (1.0%), Mineral Oil (4.5%)-emolients-Inactive ingredients-boric acid, edetate disodium, octoxynol-40, polyquaternium-1 (preservative), polysorbate 80, purified water, sodium borate decahydrate. May contain hydrochloric acid and/or sodium hydroxide (to adjust pH).</td>
		</tr>
		<tr>
			<td>Systane Complete (solution #3)</td>
			<td>Alcon Inc.</td>
			<td></td>
			<td>Propylene Glycol 0.6% Lubricant Inactive ingredients- boric acid, dimyristoyl phosphatidylglycerol, edatate disodium, hydroxypropyl guar, mineral oil, polyoxl 40 stearate, POLYQUAD&#174; (polyquaternium-1) 0.001% preservative, sorbitan tristearate, sorbitol and purified water. May contain hydrochloric acid and/or sodium hydroxide to adjust pH.</td>
		</tr>
		<tr>
			<td>TrypLE Express (cell disassociation solution)</td>
			<td>Fisher Scientific</td>
			<td><a href="https://www.thermofisher.com/order/catalog/product/12605036" target="_blank">12605036</a></td>
			<td></td>
		</tr>
	</tbody>
</table>

effect of artificial tear formulations on the metabolic activity of human corneal epithelial cells after exposure to desiccation

开发人工含脂催泪配方，通过恢复缺损的泪脂层来减少撕裂蒸发。防止细胞干燥的人工撕裂配方将产生眼部表面保护和细胞代谢活性的维持。在脱水期间，细胞经历代谢活动丧失的过程，随后细胞死亡。这项工作描述了一种评估人工撕裂制剂功效的方法。代谢染料（即阿拉马蓝）从低荧光分子雷萨祖林变为可行细胞中的荧光分子再糖素。人工撕裂制剂的生物性能被测量为配方维持细胞生存能力的能力，并且 （b） 提供细胞防干燥。生长介质和盐水用于细胞生存能力/干燥测试的控制。细胞用测试溶液孵育30分钟，然后干燥0或5分钟，在37°C和45%的相对湿度。细胞代谢活动后，初始接触和细胞干燥后，然后确定。结果表明，眼药水配方对细胞代谢活动和干燥保护的比较作用。这种方法可用于测试干眼配方，旨在用蒸发性干眼治疗个体。

一项比较三种干眼配方的研究结果见 图1。这个数字表明，这三种产品、解决方案#1、解决方案#2和解决方案 #3（材料表）对HCEC的可行性的影响存在差异。溶液#1和溶液#2对干燥前HCEC的代谢活性有显著影响。这意味着这些产品中有配方组件可以破坏 HCEC 的代谢活动。暴露在溶液中的细胞恢复18小时后细胞代谢活动进一步下降，#1表明HCEC最初受伤，18小时后损伤没有修复。相比之下，解决方案#3对HCEC的代谢活动只有轻微的影响。
图2 显示了这些含脂干眼配方对细胞保护的影响。在18小时恢复间隔内，HCEC代谢活动接近0%，溶液#1和溶液#2不能保护细胞免受干燥应激。然而，解决方案#3提供了一些防干燥压力的保护。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60812/60812fig1.jpg"> 图1:干眼脂增强产品对细胞生存能力的影响。 使用含脂干眼产品治疗后，由HCEC代谢活动测量的相对生存能力。* 与解决方案#3相比，统计学意义（p < 0.05），Δ = 相对于适当恢复期的媒体控制的统计意义（p < 0.05）。条的高度表示平均值，错误栏为 SD。 <a href="https://www.jove.com/files/ftp_upload/60812/60812fig1large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60812/60812fig2.jpg"> 图2:干眼脂增强产品对干燥保护的影响。 含脂干眼配方对细胞保护的影响。溶液#1和溶液#2不能保护细胞免受干燥压力的影响，而且随着时间的推移，它们不会从干燥效果中恢复过来。相比之下，解决方案#3提供了一些防干燥压力的保护。*=p< 0.05相对于解决方案#3，Δ=p<0.05相对于未经处理（控制）介质为适当的恢复时间。条的高度表示平均值，错误栏为 SD。 <a href="https://www.jove.com/files/ftp_upload/60812/60812fig2large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Effect of Artificial Tear Formulations on the Metabolic Activity of Human Corneal Epithelial Cells after Exposure to Desiccation at JoVE.com

Effect of Artificial Tear Formulations on the Metabolic Activity of Human Corneal Epithelial Cells after Exposure to Desiccation

本协议的目的是评估不同的人工撕裂配方是否能使用体外模型保护人类角膜上皮细胞免受干燥。在接触人工撕裂配方和干燥后，对人体角膜上皮细胞进行代谢活性评估。

人工撕裂制剂对暴露于干燥后人类角膜上皮细胞代谢活动的影响

Artificial lipid-containing tear formulations are developed to reduce tear evaporation by the restoration of a deficient tear lipid layer. Artificial tear ...

effect-artificial-tear-formulations-on-metabolic-activity-human

Research

JoVE Journal

Medicine

3.8K 次观看.  Alcon Vision, LLC. 本协议的目的是评估不同的人工撕裂配方是否能使用体外模型保护人类角膜上皮细胞免受干燥。在接触人工撕裂配方和干燥后，对人体角膜上皮细胞进行代谢活性评估。

视频: Effect of Artificial Tear Formulations on the Metabolic Activity of Human Corneal Epithelial Cells after Exposure to Desiccation

Isolation of Mouse Respiratory Epithelial Cells and Exposure to Experimental Cigarette Smoke at Air Liquid Interface

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface (ALI) as a model of differentiated respiratory epithelium. A protocol is described for isolating and exposing these cells to mainstream cigarette smoke (CS), in order to study epithelial cell responses to CS exposure. The protocol consists of three parts: the isolation of airway epithelial cells from mouse trachea, the culturing of these cells at air-liquid interface (ALI) as fully differentiated epithelial cells, and the delivery of calibrated mainstream CS to these cells in culture. The ALI culture system allows the culture of respiratory epithelia under conditions that more closely resemble their physiological setting than ordinary liquid culture systems. The study of molecular and lung cellular responses to CS exposure is a critical component of understanding the impact of environmental air pollution on human health. Research findings in this area may ultimately contribute towards understanding the etiology of chronic obstructive pulmonary disease (COPD), and other tobacco-related diseases, which represent major global health problems.

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface as a model of differentiated respiratory epithelium. A protocol is described for isolating, culturing and exposing these cells to mainstream cigarette smoke, in order to study molecular responses to this environmental toxin.

小鼠呼吸道上皮细胞和实验香烟烟雾暴露在空气分离液界面

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface (ALI) as a model of differentiated ...

科研

医学

Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells

Progress in prostate cancer research is severely limited by the availability of human-derived and hormone-na&iuml;ve model systems, which limit our ability to understand genetic and molecular events underlying prostate disease initiation. Toward developing better model systems for studying human prostate carcinogenesis, we and others have taken advantage of the unique pro-prostatic inductive potential of embryonic rodent prostate stroma, termed urogenital sinus mesenchyme (UGSM). When recombined with certain pluripotent cell populations such as embryonic stem cells, UGSM induces the formation of normal human prostate epithelia in a testosterone-dependent manner. Such a human model system can be used to investigate and experimentally test the ability of candidate prostate cancer susceptibility genes at an accelerated pace compared to typical rodent transgenic studies. Since Human embryonic stem cells (hESCs) can be genetically modified in culture using inducible gene expression or siRNA knock-down vectors prior to tissue recombination, such a model facilitates testing the functional consequences of genes, or combinations of genes, which are thought to promote or prevent carcinogenesis.
The technique of isolating pure populations of UGSM cells, however, is challenging and learning often requires someone with previous expertise to personally teach. Moreover, inoculation of cell mixtures under the renal capsule of an immunocompromised host can be technically challenging. Here we outline and illustrate proper isolation of UGSM from rodent embryos and renal capsule implantation of tissue mixtures to form human prostate epithelium. Such an approach, at its current stage, requires in vivo xenografting of embryonic stem cells; future applications could potentially include in vitro gland formation or the use of induced pluripotent stem cell populations (iPSCs).

To unravel the earliest molecular mechanisms underlying prostate cancer initiation, novel and innovative human model systems and approaches are desperately needed. The potential of pre-prostatic urogenital sinus mesenchyme (UGSM) to induce pluripotent stem cell populations to form human prostate epithelium is a powerful experimental tool in prostate research.

人类前列腺上皮细胞形成的使用鼠类泌尿生殖窦间质细胞和人类干细胞的组织重组

Progress in prostate cancer research is severely  limited by the availability of human-derived and hormone-na&iuml;ve model systems,  which limit our ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

分离，冻存和文化的人羊膜上皮细胞的临床应用

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Systems Biology of Metabolic Regulation by Estrogen Receptor Signaling in Breast Cancer

With the advent of the -omics approaches our understanding of the chronic diseases like cancer and metabolic syndrome has improved. However, effective mining of the information in the large-scale datasets that are obtained from gene expression microarrays, deep sequencing experiments or metabolic profiling is essential to uncover and then effectively target the critical regulators of diseased cell phenotypes. Estrogen Receptor &#945; (ER&#945;) is one of the master transcription factors regulating the gene programs that are important for estrogen responsive breast cancers. In order to understand to role of ER&#945; signaling in breast cancer metabolism we utilized transcriptomic, cistromic and metabolomic data from MCF-7 cells treated with estradiol. In this report we described generation of samples for RNA-Seq, ChIP-Seq and metabolomics experiments and the integrative computational analysis of the obtained data. This approach is useful in delineating novel molecular mechanisms and gene regulatory circuits that are regulated by a particular transcription factor which impacts metabolism of normal or diseased cells.

Integration of data from genome-wide sequencing experiments and metabolomics experiments is a challenge. In this paper we report, for the first time, generation, analysis and integration of transcriptome, cistrome and metabolome data from breast cancer cells treated with estradiol.

乳腺癌代谢调控的系统生物学雌激素受体信号

With the advent of the -omics approaches our understanding of the chronic diseases like cancer and metabolic syndrome has improved. However, effective ...

Turbidimetry on Human Washed Platelets: The Effect of the Pannexin1-inhibitor Brilliant Blue FCF on Collagen-induced Aggregation

Turbidimetry is a laboratory technique that is applied to measure the aggregation of platelets suspended in either plasma (platelet-rich plasma, PRP) or in buffer (washed platelets), by the use of one or a combination of agonists. The use of washed platelets separated from their plasma environment and in the absence of anticoagulants allows for studying intrinsic platelet properties. Among the large panel of agonists, arachidonic acid (AA), adenosine di-phosphate (ADP), thrombin and collagen are the most frequently used. The aggregation response is quantified by measuring the relative optical density (OD) over time of platelet suspension under continuous stirring. Platelets in homogeneous suspension limit the passage of light after the addition of an agonist, platelet shape change occurs producing a small transitory increase in OD. Following this initial activation step, platelet clots form gradually, allowing the passage of light through the suspension as a result of decreased OD. The aggregation process is ultimately expressed as a percentage, compared to the OD of platelet-poor plasma or buffer. Rigorous calibration is thus essential at the beginning of each experiment. As a general rule: calibration to 0% is set by measuring the OD of a non-stimulated platelet suspension while measuring the OD of the suspension medium containing no platelets represents a value of 100%. Platelet aggregation is generally visualized as a real-time aggregation curve. Turbidimetry is one of the most commonly used laboratory techniques for the investigation of platelet function and is considered as the historical gold standard and used for the development of new pharmaceutical agents aimed at inhibiting platelet aggregation. Here, we describe detailed protocols for 1) preparation of human washed platelets and 2) turbidimetric analysis of collagen-induced aggregation of human washed platelets pretreated with the food dye Brilliant Blue FCF that was recently identified as an inhibitor of Pannexin1 (Panx1) channels.

We describe a straightforward method for the isolation of washed platelets from human blood followed by agonist-induced platelet aggregation measurements by turbidimetry. As an example we apply this method for studying the aggregation response of human platelets to collagen after a pre-incubation with the Pannexin1 channel inhibitor Brilliant Blue FCF.

比浊法对人洗涤血小板:所述Pannexin1抑制剂的影响亮蓝FCF对胶原诱导的聚集

Turbidimetry is a laboratory technique that is applied to measure the aggregation of platelets suspended in either plasma (platelet-rich plasma, PRP) or ...

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

The human placenta is highly inaccessible for research while still in utero. The current understanding of human placental physiology in vivo is therefore largely based on animal studies, despite the high diversity among species in placental anatomy, hemodynamics and duration of the pregnancy. The vast majority of human placenta studies are ex vivo perfusion studies or in vitro trophoblast studies. Although in vitro studies and animal models are essential, extrapolation of the results from such studies to the human placenta in vivo is uncertain. We aimed to study human placenta physiology in vivo at term, and present a detailed protocol of the method. Exploiting the intraabdominal access to the uterine vein just before the uterine incision during planned cesarean section, we collect blood samples from the incoming and outgoing vessels on the maternal and fetal sides of the placenta. When combining concentration measurements from blood samples with volume blood flow measurements, we are able to quantify placental and fetal uptake and release of any compound. Furthermore, placental tissue samples from the same mother-fetus pairs can provide measurements of transporter density and activity and other aspects of placental functions in vivo. Through this integrative use of the 4-vessel sampling method we are able to test some of the current concepts of placental nutrient transfer and metabolism in vivo, both in normal and pathological pregnancies. Furthermore, this method enables the identification of substances secreted by the placenta to the maternal circulation, which could be an important contribution to the search for biomarkers of placenta dysfunction.

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

We present a detailed method to study human placental physiology in vivo at term. The method combines blood sampling from the incoming and outgoing vessels on the maternal and fetal sides of the placenta with ultrasound measurements of volume blood flow and placental tissue sampling.

人体胎盘生理学综合研究的4血管取样方法<em&gt;体内</em

The human placenta is highly inaccessible for research while still in utero. The current understanding of human placental physiology in vivo is therefore ...

Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans

Toxicological evaluation is crucial for understanding the effects of chemicals on living organisms in basic and applied biological science fields. A non-mammalian soil round worm, Caenorhabditis elegans, is a valuable model organism for toxicology studies due to its convenience and lack of animal ethics issues compared with mammalian animal systems. In this protocol, a detailed procedure of toxicological evaluation of chemicals in C. elegans is described. A clinical anticancer drug, etoposide, which targets human topoisomerase II and inhibits DNA replication of human cancer cells, was selected as a model testing chemical. Age-synchronized C. elegans eggs were exposed to either dimethyl sulfoxide (DMSO) or etoposide, and then the growth of C. elegans was monitored every day for 4 days by the stereo microscope observation. The total number of eggs laid from C. elegans treated with DMSO or etoposide was also counted by using the stereo microscope. Etoposide treatment significantly affected the growth and reproduction of C. elegans. By comparison of the total number of eggs laid from worms with different treatment periods of chemicals, it can be decided that the reproductive toxicity of chemicals on C. elegans reproduction is reversible or irreversible. These protocols may be helpful for both the development of various drugs and risk assessment of environmental toxicants.

Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans

A basic protocol to evaluate the toxicity of chemicals in a model animal, Caenorhabditis elegans, is described. The method is convenient and useful for the development of pharmaceuticals as well as for the risk assessment of various environmental pollutants.

测定化学物质对线虫线虫生长和繁殖的影响

Toxicological evaluation is crucial for understanding the effects of chemicals on living organisms in basic and applied biological science fields. A ...

A Primary Human Trophoblast Model to Study the Effect of Inflammation Associated with Maternal Obesity on Regulation of Autophagy in the Placenta

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be attributed to, in part, the dysregulation of autophagy. Aberrant changes in the expression of autophagy regulators in the placentas from obese pregnancies may be regulated by inflammatory processes associated with both obesity and pregnancy. Described here is a protocol for sampling of villous tissue and isolation of villous cytotrophoblasts from the term human placenta for primary cell culture. This is followed by a method for simulating the inflammatory milieu in the obese intrauterine environment by treating primary trophoblasts from lean pregnancies with tumor necrosis factor alpha (TNF&#945;), a proinflammatory cytokine that is elevated in obesity and in pregnancy. Through the implementation of the protocol described here, it is found that exposure to exogenous TNF&#945; regulates the expression of Rubicon, a negative regulator of autophagy, in trophoblasts from lean pregnancies with female fetuses. While a variety of biological factors in the obese intrauterine environment maintain the potential to modulate critical pathways in trophoblasts, this ex vivo system is especially useful for determining if expression patterns observed in vivo in human placentas with maternal obesity are a direct result of TNF&#945; signaling. Ultimately, this approach affords the opportunity to parse out the regulatory and molecular implications of inflammation associated with maternal obesity on autophagy and other critical cellular pathways in trophoblasts that have the potential to impact placental function.

Presented here is a protocol for sampling of human placental villous tissue followed by isolation of cytotrophoblasts for primary cell culture. Treatment of trophoblasts with TNF&#945; recapitulates inflammation in the obese intrauterine environment and facilitates the discovery of molecular targets regulated by inflammation in placentas with maternal obesity.

人体滋养层模型研究母体肥胖炎症对胎盘自噬的调节作用

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be ...

A Murine Model of Fetal Exposure to Maternal Inflammation to Study the Effects of Acute Chorioamnionitis on Newborn Intestinal Development

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing enterocolitis (NEC). However, a mechanistic link between these two conditions remains yet to be discovered. We have adopted a murine model of chorioamnionitis involving lipopolysaccharide (LPS)-induced fetal exposure to maternal inflammation (FEMI). This model of FEMI induces a sterile maternal, placental, and fetal inflammatory cascade, which is also present in many cases of clinical chorioamnionitis. Although models exist that utilize live bacteria and more accurately mimic the pathophysiology of an ascending infection resulting in chorioamnionitis, these methods may cause indirect effects on development of the immature intestinal tract and the associated developing microbiome. Using this protocol, we have demonstrated that LPS-induced FEMI results in a dose-dependent increase in pregnancy loss and preterm birth, as well as disruption of normal intestinal development in offspring. Further, we have demonstrated that FEMI significantly increases intestinal injury and serum cytokines in offspring, while simultaneously decreasing goblet and Paneth cells, both of which provide a first line of innate immunity against intestinal inflammation. Although a similar model of LPS-induced FEMI has been used to model the association between chorioamnionitis and subsequent abnormalities of the central nervous system, to our knowledge, this protocol is the first to attempt to elucidate a mechanistic link between chorioamnionitis and later perturbations in intestinal development as a potential link between chorioamnionitis and NEC.

We developed a model of chorioamnionitis to simulate fetal exposure to maternal inflammation (FEMI) without complications of live organisms to examine the effects of FEMI on development of the offspring&#8217;s intestinal tract. This allows for study of mechanistic causes for development of intestinal injury following chorioamnionitis.

胎儿接触产妇炎症的穆林模型，研究急性胆管炎对新生儿肠道发育的影响

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing ...

Electroporation-Based Genetic Modification of Primary Human Pigment Epithelial Cells Using the Sleeping Beauty Transposon System

Our increasingly aging society leads to a growing incidence of neurodegenerative diseases. So far, the pathological mechanisms are inadequately understood, thus impeding the establishment of defined treatments. Cell-based additive gene therapies for the increased expression of a protective factor are considered as a promising option to medicate neurodegenerative diseases, such as age-related macular degeneration (AMD). We have developed a method for the stable expression of the gene encoding pigment epithelium-derived factor (PEDF), which is characterized as a neuroprotective and anti-angiogenic protein in the nervous system, into the genome of primary human pigment epithelial (PE) cells using the Sleeping Beauty (SB) transposon system. Primary PE cells were isolated from human donor eyes and maintained in culture. After reaching confluence, 1 x 104 cells were suspended in 11 &#181;L of resuspension buffer and combined with 2 &#181;L of a purified solution containing 30 ng of hyperactive SB (SB100X) transposase plasmid and 470 ng of PEDF transposon plasmid. Genetic modification was carried out with a capillary electroporation system using the following parameters: two pulses with a voltage of 1,100 V and a width of 20 ms. Transfected cells were transferred into culture plates containing medium supplemented with fetal bovine serum; antibiotics and antimycotics were added with the first medium exchange. Successful transfection was demonstrated in independently performed experiments. Quantitative polymerase chain reaction (qPCR) showed the increased expression of the PEDF transgene. PEDF secretion was significantly elevated and remained stable, as evaluated by immunoblotting, and quantified by enzyme-linked immunosorbent assay (ELISA). SB100X-mediated transfer allowed for a stable PEDF gene integration into the genome of PE cells and ensured the continuous secretion of PEDF, which is critical for the development of a cell-based gene addition therapy to treat AMD or other retinal degenerative diseases. Moreover, analysis of the integration profile of the PEDF transposon into human PE cells indicated an almost random genomic distribution.

We have developed a protocol to transfect primary human pigment epithelial cells by electroporation with the gene encoding pigment epithelium-derived factor (PEDF) using the Sleeping Beauty (SB) transposon system. Successful transfection was demonstrated by quantitative polymerase chain reaction (qPCR), immunoblotting, and enzyme-linked immunosorbent assay (ELISA).