Name: isolation of retinal pigment epithelial cells from guinea pig eyes
Uploaded: 2023-05-09T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Retinal Pigment Epithelial Cells from Guinea Pig Eyes at JoVE.com

这项研究得到了日本科学促进会海外研究奖学金（S.G.），Loris和David Rich博士后学者（S.G.）以及国立卫生研究院国家眼科研究所（R01EY012392;C.F.W.）。

本研究中使用的所有动物护理和治疗均符合ARVO关于在眼科和视力研究中使用动物的声明。实验方案得到了加州大学伯克利分校动物护理和使用委员会的批准。
1.豚鼠眼摘除术
<ol><li>用心内注射在麻醉下递送的戊巴比妥钠（5%异氟醚氧溶液）对豚鼠实施安乐死。</li>
	<li>借助镊子和剪刀将眼睛去核，并立即将它们转移到带有无菌磷酸盐缓冲盐水（PBS）的10厘米培养?...

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+vascular+patterns+in+domestic+animals.">Retinal vascular patterns in domestic animals.</a> Research in Veterinary Science. 47 (1), 34-42 (1989).</li><li>Araki, H., 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=Base-resolution+methylome+of+retinal+pigment+epithelial+cells+used+in+the+first+trial+of+human+induced+pluripotent+stem+cell-based+autologous+transplantation.">Base-resolution methylome of retinal pigment epithelial cells used in the first trial of human induced pluripotent stem cell-based autologous transplantation.</a> Stem Cell Reports. 13 (4), 761-774 (2019).</li><li>Sonoda, S., 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=A+protocol+for+the+culture+and+differentiation+of+highly+polarized+human+retinal+pigment+epithelial+cells.">A protocol for the culture and differentiation of highly polarized human retinal pigment epithelial cells.</a> Nature Protocols. 4 (5), 662-673 (2009).</li><li>Fernandez-Godino, R., Garland, D. 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在本文中，我们描述了一种从年轻的色素有色豚鼠的眼睛中分离RPE的方法，适用于RPE基因表达分析。该协议的优点是它产生相对不受污染的高质量RPE样品，RNA适合RNA特异性分析，但相对简单有效。虽然在这里提供的示例中，RPE样本是从年轻（2周大）豚鼠的眼睛收集的，但该方案也已成功用于从老年（年轻成年）动物中收集RPE样本。
对于眼科手术或解剖经验很少的研究人员来?...

RPE包括位于神经视网膜和血管脉络膜之间的独特单层色素细胞，RPE在正常视网膜功能的发育和维持（包括光转导1，2）方面具有公认的作用。最近，RPE在眼睛生长调节3中被赋予了额外的关键作用，因此，近视4的发展。该分配基于RPE的关键位置，位于视网膜和脉络膜之间，以及现在广泛接受的眼睛生长以及因此屈光不正在局部调节5。RPE被认为作为信号中继起着关键作用，将视网膜（生长调节信号的假定来源）连接到脉络膜和巩膜，这是中继信号的两个目标6，7，8。
大多数近视的特征是轴向长度的增加不能被认为是良性的，涉及视网膜、脉络膜和/或巩膜的病理生理变化代表了不可避免的，现在公认的眼部过度伸长的后果7，9。在这种情况下，RPE可能是最脆弱的，因为作为非有丝分裂组织，它只能通过单个细胞的拉伸和变薄来容纳膨胀的玻璃体室。虽然RPE在近视相关病变（如近视性黄斑变性）中的作用尚未完全了解，但RPE与许多其他视网膜疾病的发病机制有关，包括地理萎缩，这是失明的主要原因之一，与记录的视网膜异常有关，RPE和脉络膜10，11，12.
成功分离RPE细胞，不受邻近眼组织的污染，可能开辟了许多研究机会，以获得对各种眼睛/视网膜疾病机制的新见解。然而，RPE的分离已被证明具有挑战性，许多已发表的研究因此使用组合视网膜/ RPE或RPE /脉络膜样本13，14，15。涉及成功分离适合分子生物学研究的质量的RPE的研究仅限于小鸡和小鼠眼睛16，17。例如，Wang等人描述的同步RPE分离和RNA稳定（SRIRS）方法18。在小鼠中分离RPE细胞似乎在豚鼠眼睛中效果不佳。这里描述的方案代表了一种方法的改进，该方法最初由其中一位作者（M.F.）用树鼩眼制作原型，并已被证明可以从年轻色素豚鼠的眼睛中产生高质量的RPE样品，适用于RNA和其他分子生物学分析19。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL Syringe with Slip Tip</td>
			<td>Bd Vacutainer Labware Medical</td>
			<td>22-253-260</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Invitrogen</td>
			<td>21985-023</td>
			<td></td>
		</tr>
		<tr>
			<td>6 Well Tissue Culture Plate with Lid, Flat Bottom, Sterile</td>
			<td>pectrum Chemical Mfg. Corp</td>
			<td>970-95008</td>
			<td></td>
		</tr>
		<tr>
			<td>12 Well Tissue Culture Plate with Lid, Individual, Sterile</td>
			<td>Thomas Scientific LLC</td>
			<td>1198D72</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent 2100 Bioanalyzer</td>
			<td></td>
			<td></td>
			<td>automated electrophoresis to check RNA quality</td>
		</tr>
		<tr>
			<td>Balanced Salt Solutions</td>
			<td>Gibco</td>
			<td>10010031</td>
			<td></td>
		</tr>
		<tr>
			<td>Bonn Micro Forceps, Straight Smooth, 0.3 mm Tip, 7 cm</td>
			<td>Fine Science Tools, Inc.</td>
			<td>11083-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont forceps no. 5</td>
			<td>ROBOZ</td>
			<td>RS-5045</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypodermic disposable needles</td>
			<td>Exelint International, Co.</td>
			<td>26419</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypodermic disposable needles</td>
			<td>Exelint International, Co.</td>
			<td>26437</td>
			<td></td>
		</tr>
		<tr>
			<td>MiniSpin Microcentrifuges</td>
			<td>Eppendorf</td>
			<td>540108</td>
			<td>Max. Speed: 8,000 g</td>
		</tr>
		<tr>
			<td>RNAlater Stabilization Solution</td>
			<td>Invitrogen</td>
			<td>AM7020</td>
			<td>tissue storage reagent</td>
		</tr>
		<tr>
			<td>RNeasy Mini kits</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td>RNA isolation kit</td>
		</tr>
		<tr>
			<td>Student Vannas Spring Scissors</td>
			<td>Fine Science Tools, Inc.</td>
			<td>91500-09</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of retinal pigment epithelial cells from guinea pig eyes

该协议描述了从年轻色素豚鼠的眼睛中分离视网膜色素上皮（RPE）的细胞，以便在分子生物学研究中的潜在应用，包括基因表达分析。在眼睛生长调节和近视的背景下，RPE可能充当生长调节信号的细胞中继，因为它位于视网膜和眼睛的两壁（如脉络膜和巩膜）之间。虽然已经为小鸡和小鼠开发了分离RPE的方案，但这些方案已被证明不能直接转化为豚鼠，豚鼠已成为一种重要且广泛使用的哺乳动物近视模型。在这项研究中，分子生物学工具用于检查特定基因的表达，以确认样品没有来自邻近组织的污染。该方案的价值已经在暴露于近视诱导光学离焦的年轻色素豚鼠的 RPE 的 RNA-Seq 研究中得到证明。除了眼睛生长调节之外，该协议在视网膜疾病的研究中还有其他潜在的应用，包括近视性黄斑病变，这是近视失明的主要原因之一，其中与RPE有关。该技术的主要优点是相对简单，一旦完善，即可产生适合分子生物学研究（包括RNA分析）的高质量RPE样品。

使用上述方案收集的RPE样品的分析显示保存良好的RNA（RIN >8.0，图2B），每只眼睛240.2 ng±35.1 ng（n = 8，NanoDrop，图2B）。 为了进一步评估分离的RPE样品的质量，特别是没有脉络膜和巩膜污染物，我们检查了RPE样品中每种组织的代表性基因的表达19。与脉络膜和巩膜中的Rpe65表达水平相比，RPE样品显示出Rpe65（RPE特异性基因）的表达...

Watch this Scientific Journal Video about Isolation of Retinal Pigment Epithelial Cells from Guinea Pig Eyes at JoVE.com

Isolation of Retinal Pigment Epithelial Cells from Guinea Pig Eyes

我们描述了一种从年轻色素豚鼠的眼睛中分离视网膜色素上皮（RPE）细胞的简单有效的方法。该程序允许对分离的RPE进行后续分子生物学研究，包括基因表达分析。

从豚鼠眼睛中分离视网膜色素上皮细胞

This protocol describes the isolation of cells of the retinal pigment epithelium (RPE) from the eyes of young pigmented guinea pigs for potential ...

视网膜色素上皮或RPE对视网膜健康至关重要。研究孤立的RPE对于视网膜疾病至关重要。这种从豚鼠中分离RPE的方法，一种流行的近视模型，有助于解决具有挑战性的问题。该技术的主要优点是它相对简单，并且可以产生适合生物分子研究（包括RPE分析）的高质量RPE样品。通过完美地遵循所展示的技术并观察如何操作解剖工具，人们可以通过一些练习快速掌握此过程。在人道地对豚鼠实施安乐死并摘除动物的眼睛后，立即将眼睛转移到含有无菌磷酸盐缓冲盐水或PBS的10厘米培养皿中清洗眼睛。清洗后，将眼睛转移到新鲜的PBS溶液中。现在在解剖显微镜下工作，使用18号针在一只眼睛的巩膜上开一个初始的小开口，在角膜和巩膜之间的角膜缘边界后面约一毫米处。使用剪刀去除前段，包括角膜、虹膜、睫状体和晶状体。然后处理剩余的后眼段，用镊子抓住并轻轻拉扯Zinn的带状体，然后将视网膜从RPE/脉络膜/巩膜复合体中分离，并剥离视网膜而不碎裂。视网膜完全移除后，将剩余的后眼杯（包括 RPE、脉络膜和巩膜）浸入含有 2 毫升组织储存试剂的 12 孔板的一个孔中，并保持浸泡五分钟。要冲洗掉储存试剂，请将眼罩转移到另一个装有四毫升PBS的孔中10秒钟，然后将其移至装有两毫升PBS的第三个孔中。在进行最后的RPE隔离步骤之前，准备一个装满PBS的一毫升注射器，并连接一个30号针头。轻轻推动注射器柱塞以产生PBS喷射流。在显微镜下工作，首先，将PBS流对准RPE，在其上留下一个小的撕裂或孔。然后将PBS流引导到创建的开口中，将RPE作为片材从脉络膜中分离。将RPE从脉络膜上分离后，将RPE细胞收集在无针一毫升注射器中，并将收集的样品转移到1.5毫升管中。用收集的RPE以8， 000 x g 离心管一分钟以获得RPE沉淀。对于用于RNA分析的样品，丢弃上清液，即PBS溶液，并用RNA分离试剂盒中包含的350毫升裂解缓冲液代替。上下移液内容物 20 次，以充分混合并保持样品质量。为了长期储存和保存，将样品转移到零下80摄氏度的冰箱中。在通过电泳评估样品质量之前，请使用 RNA 分离试剂盒并按照制造商的说明从 RPE 样品中分离和收集 RNA。在我们的研究中，收集的RPE样品显示出保存完好的RNA，其RNA完整性数或RIN大于8。每只眼睛的总RNA计数约为240纳克。发现RPE特异性基因Rpe65的表达水平在RPE样品中明显高于脉络膜和巩膜。相比之下，RPE样品显示出所选脉络膜巩膜特异性基因α-one型的最小表达，表明分离的RPE样品中没有脉络膜和巩膜污染物。该程序最重要的步骤是顺利去除视网膜，而不会留下任何视网膜碎片。这种RPE分离程序需要解释体外细胞培养研究，但存在一些重要差异，包括分离后浸没RPE细胞的培养基的选择。

Research

JoVE Journal

Biology

Isolation of Retinal Stem Cells from the Mouse Eye

从鼠眼视网膜干细胞的分离

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

科研

生物学

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

人类iPS细胞的微小RNA表达谱，视网膜色素上皮衍生自IPS和胎儿视网膜色素上皮

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, ...

Subretinal Transplantation of Human Embryonic Stem Cell Derived-retinal Pigment Epithelial Cells into a Large-eyed Model of Geographic Atrophy

人胚胎干细胞源性视网膜色素上皮细胞移植视网膜大眼模型的地理萎缩

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, ...

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

小鼠视网膜色素上皮细胞的原代培养

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the ...

Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy

哺乳动物原色素上皮细胞的分离、培养和基因工程用于非病毒基因治疗

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients &#62;60 years, affecting ~30 million people worldwide. AMD is a ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

原发性小鼠视网膜色素上皮细胞的有效解剖与培养

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...

Isolation of Epithelial Cells from Human Dental Follicle

从人牙囊中分离上皮细胞

The dental follicle (DF) was harvested during the removal of an impacted third molar by an oral maxillofacial surgeon. Epithelial cell isolation was ...

Primary Culture of Porcine Retinal Pigment Epithelial Cells

猪视网膜色素上皮细胞原代培养

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. ...

Efficient and Consistent Generation of Retinal Pigment Epithelium/Choroid Flatmounts from Human Eyes for Histological Analysis

从人眼高效、一致地生成视网膜色素上皮/脉络膜平面安装，用于组织学分析

The retinal pigment epithelium (RPE) and retina are functionally and structurally connected tissues that work together to regulate light perception and ...

Real-Time Analysis of Bioenergetics in Primary Human Retinal Pigment Epithelial Cells Using High-Resolution Respirometry

使用高分辨率呼吸测定法实时分析原代人视网膜色素上皮细胞中的生物能量学

Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration ...