改进脂褐素模型和高极化人视网膜色素上皮培养物中外段吞噬能力的定量
Summary
该协议描述了高度分化和极化的人视网膜色素上皮（RPE）培养物中的脂褐素积累模型和改进的外段（OS）吞噬测定，以检测RPE的总OS消耗/降解能力。这些方法克服了以前的脂褐素模型和经典的脉搏追逐外段吞噬测定的局限性。
Abstract
视网膜色素上皮（RPE）对感光器外段的日常吞噬作用有助于细胞内衰老色素（称为脂褐素）的积累。脂褐素的毒性在斯塔加特病（最常见的遗传性视网膜变性）中已得到充分证实，但在年龄相关性黄斑变性（AMD）中更具争议性，AMD是发达国家不可逆失明的主要原因。确定人类脂褐素的毒性一直很困难，斯塔加特的动物模型毒性有限。因此，需要模拟人类体内RPE的体外模型来更好地了解脂褐素的产生，清除和毒性。迄今为止，大多数细胞培养脂褐素模型都在细胞系中，或者涉及向RPE喂食复杂脂褐素混合物的单一组分，而不是整个感光器外段的片段/尖端，从而产生更完整和生理性的脂褐素模型。这里描述的是一种诱导脂褐素样物质（称为不可消化自发荧光材料，或UAM）在高度分化的原代人产前RPE（hfRPE）和诱导多能干细胞（iPSC）衍生RPE中积累的方法。UAM通过重复喂食由RPE通过吞噬作用吸收的紫外线处理的OS片段在培养物中积累。还讨论了UAM在体内与脂褐素近似和不同的关键方法。在这种脂褐素样积累模型的同时，介绍了用于区分UAM颗粒的宽自发荧光光谱与并发抗体染色的成像方法。最后，为了评估UAM对RPE吞噬能力的影响，引入了一种量化外段片段/尖端摄取和分解的新方法。该方法被称为“总消耗容量”，克服了经典外段“脉冲追逐”测定中固有的RPE吞噬能力的潜在误解。这里介绍的模型和技术可用于研究脂褐素的生成和清除途径以及推定的毒性。
Introduction
视网膜色素上皮（RPE）为上覆的光感受器提供关键支持，包括感光器外段尖端或片段的日常摄取和降解（在整个协议中，缩写OS代表OS尖端或片段，而不是整个外段）。这种对有丝分裂后RPE的每日摄取最终使吞噬溶酶体容量过载，并导致难以消化的自发荧光细胞内物质（称为脂褐素）的积聚。有趣的是，一些研究还表明，RPE脂褐素可以在没有OS吞噬作用的情况下积累1，2。脂褐素具有许多成分，包括源自视觉周期类视黄醇的交联加合物，对于80岁以上的人来说，可以占据RPE细胞体积的近20%3。
脂褐素是否有毒一直备受争议。斯塔加特病是光感受器和RPE的常染色体隐性变性，其中ABCA4的突变触发了光感受器外段内含有的视觉周期类视黄醇的不当处理。不正确的类视黄醇加工会导致异常交联和双类视黄醇物种的形成，包括双类视黄醇 N-视黄亚基亚基-N-视黄基乙醇胺 （A2E）。研究表明A2E毒性的多种机制4，5。脂褐素有助于临床成像期间眼底自发荧光信号，Stargardt 的患者和动物模型均在视网膜变性前显示眼底自发荧光增加，表明脂褐素水平与毒性之间存在相关性6，7。然而，随着年龄的增长，脂褐素会在所有人体内积累而不会引发RPE变性。此外，在年龄相关性黄斑变性（AMD）中，RPE变性仅发生在老年患者中，具有早期和中期形式的疾病的患者比年龄匹配的非患病人类具有更少的眼底自发荧光信号8。这些临床发现已在组织学水平上得到验证9，10。
RPE脂褐素积累的动物模型也对脂褐素的毒性留下了一些模糊性。ABCA4敲除小鼠在色素背景上不显示视网膜变性，而在白化背景或暴露于蓝光11，12时则显示视网膜变性。此外，通过ABCA4敲除产生的脂褐素的毒性可能与自然衰老时发生的更缓慢积累的脂褐素不同，如AMD13所示。
脂褐素积累的体外模型为研究脂褐素积累对RPE健康的影响提供了一种替代方案。这种模型允许操纵脂褐素成分，从喂养单个类视黄醇成分到喂养OS，并允许在人类而不是动物RPE中进行研究。在过去的几十年中，已经开发了多种方法来模拟培养中的RPE脂褐素。与其他小组一起，Boulton博士的小组每天通过4至7个来自4至85岁14岁供体的人类原代RPE细胞喂食牛OS长达三个月。或者，自噬的抑制也导致脂褐素在第3至7代原代人RPE培养物中积累15。然而，在高度分化的传代1代原代人产前RPE（hfRPE）培养物中，亚致死性溶酶体抑制未能诱导脂褐素，即使每天重复添加OS16。
作为一种更还原的方法，其他人将单一脂褐素成分喂入培养物中，尤其是双类维生素A2E4，17。这些研究很有价值，因为它们定义了单个脂褐素成分的潜在直接毒性机制，例如涉及溶酶体胆固醇和神经酰胺稳态18。同时，关于A2E19的毒性存在争议，将其直接喂给细胞绕过脂褐素积累的典型途径，这涉及光感受器OS的吞噬作用。为了将脂褐素的所有成分输送到RPE培养物中，Boulton和Marshall从人眼中纯化了脂褐素，并将其喂入传代4至7来自胎儿和老年人类供体的人类原代RPE培养物20。虽然具有创新性，但该方法代表了重复实验的有限脂褐素来源。
虽然在许多系统中重复喂食 OS 到 RPE 培养物会产生脂褐素，但在高度分化的原代 RPE 培养物中却无法做到这一点16。光氧化OS诱导交联反应，如在体内脂褐素形成过程中自然发生的双类视黄醇形成。这可以加速RPE培养系统中脂褐素样颗粒的形成，即使是那些高度分化且抗脂褐素积累的颗粒16。在这里，介绍了一种诱导脂褐素样颗粒在高度分化的hfRPE和人iPSC-RPE中积累的方法，该方法根据Wihlmark发表的方案21进行了修改。该方法的优点是使用与体内脂褐素发生相同的来源（光感受器OS）和途径（吞噬溶酶体OS摄取）诱导脂褐素样颗粒。此外，它是在高度分化并在多项研究中验证的人类 RPE 培养物上进行的，以在体内复制人类 RPE 22，23，24。 这些脂褐素样颗粒被称为难消化的自发荧光材料（UAM），并在该协议中提供了将UAM与体内脂褐素进行比较的数据和讨论。 除了在高度分化的人类RPE中构建和评估UAM培养物的方法外，还介绍了一种评估RPE OS吞噬作用的更新方法。已经介绍了多种用于定量OS吞噬作用的优秀脉冲追踪方法，包括蛋白质印迹、免疫细胞化学和FACS25，26，27。然而，在OS脉冲追逐的早期，导致OS摄取不良的条件可以与促进内部OS快速退化的条件混为一谈。此处介绍的方法测量 RPE 完全消耗/降级的引入操作系统的总量（“总消耗容量”），有助于消除这种歧义。预计利用这些方案对脂褐素毒性的见解，包括使用“总消耗容量”方法对OS吞噬速率的影响，将用于阐明脂褐素在体内的毒性。
Protocol
涉及获取和使用人体组织的本协议已由密歇根大学机构审查委员会（HUM00105486）审查和批准。 1.光氧化外段尖端和碎片的制备 注意：深色适应的牛视网膜是在冰上购买和运输的（见 材料表）。从这些视网膜中，按照先前发布的方案23纯化OS。 外段与紫外灯交联将聚四氟乙烯涂层载玻片（参见 材料表…
Representative Results
OS光氧化的设置如图1Ai所示。聚四氟乙烯涂层载玻片允许在每个开放的矩形中加载大量溶液中的OS，而不会扩散到载玻片的其余部分。将带有OS的载玻片包含在盖子关闭的无菌培养皿中，并将紫外灯放置在载玻片上，如图1Aii所示。或者，可以将载玻片置于UV交联剂装置中，如图1Aiii所示。光氧化后，OS自发荧光显着增加，通过显微镜…
Discussion
虽然RPE脂褐素已经研究了几十年，但其毒性存在争议2，9，16，42。鉴于动物模型11中脂褐素的毒性含糊不清，使用人类RPE的体外模型是有价值的。已经描述了一系列体外脂褐素积累模型，但没有一个模型同时利用OS喂养和高度成熟和分化的人类RPE培养物。这种组合代表了…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作部分得到了玻璃体视网膜手术基金会（VRSF），视力之战（FFS）和国际视网膜研究基金会（IRRF）的资助。J.M.L.M.目前由美国国家眼科研究所（EY033420）的K08资助。没有联邦资金用于高频交易研究。进一步的支持来自James Grosfeld Initiative for Dry AMD和以下私人捐助者：Barbara Dunn和Dee & Dickson Brown。
Materials
| 100 mm cell culture dish | Corning | #353003 | Others also work |
| 24-well Transwells | Corning | #3470 | |
| Anti-LC3 antibody | Cell Signaling Technology | #4801S | 1:1000 dilution |
| Anti-rhodopsin antibody 1D4 | Abcam | #5417 | 1:1000 dilution. Epitope is C-terminal. |
| Anti-rhodopsin antibody 4D2 | EnCor Biotech | MCA-B630 | 1:5000 dilution for western blot, 1:1000 dilution for immunostaining. Epitope is N-terminal. |
| Autofluorescence quencher | Biotium | #23007 | TrueBlack Lipofuscin Autofluorescence Quencher |
| Autofluorescence quencher | Vector Laboratories | SP-8400 | Vector TrueVIEW Autofluorescence Quenching Kit |
| Bodipy 493/503 | Life Technologies | D3922 | |
| Cholesterol esterase | Life Technologies | From A12216 kit | |
| Confocal microscope | Leica | Leica Stellaris SP8 with FALCON module | |
| Dark-adapted bovine retinas | W. L. Lawson Company | Dark-adapted bovine retinas (pre-dissected) | Contact information: https://wllawsoncompany.com/ (402) 499-3161 stacy@wllawsoncompany.com |
| Filipin | Sigma-Aldrich | F4767 | |
| Flow cytometer | Thermo Fisher | Attune NxT | |
| Flow cytometer analysis software | BD | FlowJo | |
| Handheld UV light | Analytik Jena US | UVGL-55 | |
| Human MFG-E8 | Sino Biological | 10853-H08B | |
| Human purified Protein S | Enzyme Research Laboratories | HPS | |
| Laemmli sample buffer | Thermo Fisher | J60015-AD | |
| LDH assay | Promega | J2380 | LDH-Glo Cytotoxicity Assay |
| Mounting media | Invitrogen | P36930 | Prolong Gold antifade reagent |
| Nile red | Sigma-Aldrich | #72485 | |
| Polytetrafluoroethylene-coated slides | Tekdon | Customized | Customized specifications: PTFE mask with the following "cut-outs" - 3 glass rectangles, each measuring 17 mm x 9 mm, oriented so that the 17 mm side is 4 mm from the top of the slide and 4 mm from the bottom of the slide, assuming a standard microscope slide of 25 mm x 75 mm. Each rectangle is spaced at least 6 mm away from other rectangles and the edges of the slide. Print PTFE mask on a slide with frosted glass on one side to allow for labeling of the slide. |
| Protease inhibitors | Cell Signaling Technology | #5872 | |
| Protein assay | Bio-Rad | #5000122 RC DC protein assay | |
| TEER electrode | World Precision Instruments | STX3 | |
| Trans-epithelial electrical resistance (TEER) meter | World Precision Instruments | EVOM3 | |
| Ultraviolet crosslinker device | Analytik Jena US | UVP CL-1000 |
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Cite This Article
Zhang, Q., Autterson, G., Miller, J. M. L. Improved Lipofuscin Models and Quantification of Outer Segment Phagocytosis Capacity in Highly Polarized Human Retinal Pigment Epithelial Cultures. J. Vis. Exp. (194), e65242, doi:10.3791/65242 (2023).