A Novel Method of Mouse RPE Explant Culture and Effective Introduction of Transgenes Using Adenoviral Transduction for In Vitro Studies in AMD.
A Novel Method of Mouse RPE Explant Culture and Effective Introduction of Transgenes Using Adenoviral Transduction for In Vitro Studies in AMD.
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DOI：
10.3390/ijms222111979
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发表时间：
2021-11-05
影响因子：
5.6
通讯作者：
Sinha D
中科院分区：
文献类型：
--
作者：
Shang P;Stepicheva NA;Liu H;Chowdhury O;Franks J;Sun M;Hose S;Ghosh S;Yazdankhah M;Strizhakova A;Stolz DB;Zigler JS Jr;Sinha D
视网膜色素上皮（retinal pigment epithelium，RPE）变性是老年性黄斑变性（age-related macular degeneration，AMD）最重要的表型改变之一，AMD是老年人视力丧失的主要原因。虽然培养的具有原始特性的极化RPE细胞在研究RPE生物学和遗传和/或药理学操作的后果的体外模型中是有价值的，但是建立小鼠原代PRE细胞培养物或多能干细胞衍生的RPE细胞的程序是耗时的并且产生有限数量的细胞。因此，建立小鼠原位RPE培养系统是非常可取的。在这里，我们描述了一种新的和有效的方法，RPE外植体培养，允许获得生物学相关的RPE细胞原位。这些RPE外植体（本文中称为RPE平板）在培养物中存活至少7天，可以用腺病毒构建体有效地转导，和/或用各种药物/化学品处理，然后对感兴趣的信号传导途径/生物过程进行下游分析，例如评估自噬通量、炎症反应和受体酪氨酸激酶刺激。这种RPE外植体培养方法对于RPE生物学和AMD研究领域中的药理学和机制研究是非常有益的。
Degeneration of retinal pigment epithelium (RPE) is one of the most critical phenotypic changes of age-related macular degeneration (AMD), the leading cause of vision loss in the elderly. While cultured polarized RPE cells with original properties are valuable in in vitro models to study RPE biology and the consequences of genetic and/or pharmacological manipulations, the procedure to establish mouse primary PRE cell culture or pluripotent stem cell-derived RPE cells is time-consuming and yields a limited number of cells. Thus, establishing a mouse in situ RPE culture system is highly desirable. Here we describe a novel and efficient method for RPE explant culture that allows for obtaining biologically relevant RPE cells in situ. These RPE explants (herein referred to as RPE flatmounts) are viable in culture for at least 7 days, can be efficiently transduced with adenoviral constructs, and/or treated with a variety of drugs/chemicals followed by downstream analysis of the signaling pathways/biological processes of interest, such as assessment of the autophagy flux, inflammatory response, and receptor tyrosine kinases stimulation. This method of RPE explant culture is highly beneficial for pharmacological and mechanistic studies in the field of RPE biology and AMD research.
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DOI：
10.1007/978-3-030-27378-1_64
发表时间：
2019-01-01
期刊：
RETINAL DEGENERATIVE DISEASES: MECHANISMS AND EXPERIMENTAL THERAPY
影响因子：
--
作者：
Umapathy, Ankita;Williams, David S.
通讯作者：
Williams, David S.
影响因子：
39.3
作者：
Liu T;Zhang L;Joo D;Sun SC
通讯作者：
Sun SC
影响因子：
7.5
作者：
Hazim RA;Karumbayaram S;Jiang M;Dimashkie A;Lopes VS;Li D;Burgess BL;Vijayaraj P;Alva-Ornelas JA;Zack JA;Kohn DB;Gomperts BN;Pyle AD;Lowry WE;Williams DS
通讯作者：
Williams DS
影响因子：
7.8
作者：
Shang P;Valapala M;Grebe R;Hose S;Ghosh S;Bhutto IA;Handa JT;Lutty GA;Lu L;Wan J;Qian J;Sergeev Y;Puertollano R;Zigler JS Jr;Xu GT;Sinha D
通讯作者：
Sinha D
影响因子：
6.4
作者：
Shrestha, Archana;Gohari, Iman Mehdizadeh;McClane, Bruce A.
通讯作者：
McClane, Bruce A.