Akeo K, Hiramitsu T, Kanda T, Yorifuji H, Okisaka S
Department of Ophthalmology, National Defense Medical College, Saitama, Japan.
Curr Eye Res. 1996 May;15(5):467-76. doi: 10.3109/02713689609000758.
Outer segments of the photoreceptor rods that are phagocytized by the retinal pigment epithelial (RPE) cells contain a high proportion of polyunsaturated fatty acids (PUFA). PUFA are susceptible to lipid peroxidation. We hypothesized that the resulting peroxides could injure RPE cells leading to retinal degeneration. Accordingly, we compared the effects of linoleic acid (LA) and its hydroperoxide (LHP) on the growth and morphology of RPE cells using laser scanning microscopy and transmission microscopy.
We counted the number of RPE cells after incubation for 24 and 48 hrs with concentrations of LA or LHP of 0.035, 0.175, and 0.35 mM. To observe the actin filaments, cultured RPE cells were stained with rhodamine phalloidin. The cells were prefixed with 2% glutaraldehyde and postfixed in 1% osmium tetroxide. Specimens were embedded in Epon 812 after dehydration, and the ultrathin sections were doubly stained with 2% uranyl acetate and 2% lead acetate for examination by transmission electron microscopy.
Exposure to LA or LHP produced dose-dependent damage to RPE cells with a significantly greater effects of LHP than LA. After incubation for 24 hrs with 0.35 mM LA, the number of vacuoles in RPE cells exceeded that observed in control RPE cells by 365 nm laser microscopy. Exposure to 0.35 mM LHP for 24 hrs produced a pycnotic nucleus, with diffuse and granular autofluorescences observed in and around it. Exposure of RPE cells to 0.35 mM LA for 24 hrs showed that the LA incorporated into the lysosomes was digested and released extracellularly from lysosomes via exocytotic vesicles. However, such exposure to LHP damaged the RPE cells, including the membranes in the pinocytotic vesicles. The packed membranes resembled myelin.
While the LA incorporated into the lysosomes was released extracellularly, LHP persisted in the RPE cells, being observed as autofluorescent lipofuscin-like materials. LHP was cytotoxic, and caused damage to the membranes of pinocytotic vesicles and lysosomes.
被视网膜色素上皮（RPE）细胞吞噬的光感受器视杆细胞外段含有高比例的多不饱和脂肪酸（PUFA）。PUFA易发生脂质过氧化。我们推测由此产生的过氧化物可能损伤RPE细胞，导致视网膜变性。因此，我们使用激光扫描显微镜和透射显微镜比较了亚油酸（LA）及其氢过氧化物（LHP）对RPE细胞生长和形态的影响。
我们用浓度为0.035、0.175和0.35 mM的LA或LHP孵育24小时和48小时后，对RPE细胞进行计数。为了观察肌动蛋白丝，用罗丹明鬼笔环肽对培养的RPE细胞进行染色。细胞先用2%戊二醛固定，后用1%四氧化锇固定。脱水后将标本包埋在Epon 812中，超薄切片用2%醋酸铀和2%醋酸铅双重染色，用于透射电子显微镜检查。
暴露于LA或LHP会对RPE细胞产生剂量依赖性损伤，LHP的影响明显大于LA。用0.35 mM LA孵育24小时后，通过365 nm激光显微镜观察到RPE细胞中的空泡数量超过对照RPE细胞。暴露于0.35 mM LHP 24小时会导致细胞核固缩，在其内部和周围观察到弥漫性和颗粒状自发荧光。RPE细胞暴露于0.35 mM LA 24小时显示，掺入溶酶体的LA被消化，并通过胞吐小泡从溶酶体释放到细胞外。然而，这种暴露于LHP会损伤RPE细胞，包括胞饮小泡中的膜。堆积的膜类似于髓磷脂。
虽然掺入溶酶体的LA释放到细胞外，但LHP在RPE细胞中持续存在，表现为自发荧光的脂褐素样物质。LHP具有细胞毒性，会对胞饮小泡和溶酶体的膜造成损伤。
