Bascom Palmer Eye Institute, Miami Integrative Metabolomics Research Center, University of Miami, Miami, FL, USA.
Department of Ophthalmology, Bascom Palmer Eye Institute, Miller School of Medicine at University of Miami, Miami, FL, USA.
Methods Mol Biol. 2024;2816:175-191. doi: 10.1007/978-1-0716-3902-3_17.
The trabecular meshwork (TM) from primary open-angle glaucoma (POAG) cases has been found to contain decreased levels of intracellular plasmalogens. Plasmalogens are a subset of lipids involved in diverse cellular processes such as intracellular signaling, membrane asymmetry, and protein regulation. Proper plasmalogen biosynthesis is regulated by rate-limiting enzyme fatty acyl-CoA reductase (Far1). ATPase phospholipid transporting 8B2 (ATP8B2) is a type IV P-type ATPase responsible for the asymmetric distribution of plasmalogens between the intracellular and extracellular leaflets of the plasma membranes. Here we describe the methodology for extraction and culturing of TM cells from corneal tissue and subsequent downregulation of ATP8B2 using siRNA transfection. Further quantification and downstream effects of ATP8B2 gene knockdown will be analyzed utilizing immunoblotting techniques.
已发现原发性开角型青光眼（POAG）病例的小梁网（TM）中细胞内血浆质减少。血浆质是参与多种细胞过程的脂质亚类，如细胞内信号转导、膜不对称性和蛋白质调节。适当的血浆质生物合成受限速酶脂肪酸 CoA 还原酶（Far1）调节。ATP 酶磷脂转运蛋白 8B2（ATP8B2）是一种 IV 型 P 型 ATP 酶，负责将血浆质在质膜的细胞内和细胞外叶之间不对称分布。在这里，我们描述了从角膜组织中提取和培养 TM 细胞的方法，以及使用 siRNA 转染下调 ATP8B2 的方法。进一步利用免疫印迹技术分析 ATP8B2 基因敲低的定量和下游效应。
