Cyclophilin D在氧化应激下人视网膜色素上皮细胞中的表达

目的　检测氧化应激下人视网膜色素上皮（ｒｅｔｉｎａｌｐｉｇｍｅｎｔｅｐｉｔｈｅｌｉｕｍ，ＲＰＥ）细胞内ＣｙｃｌｏｐｈｉｌｉｎＤ的表达，初步探索ＲＰＥ细胞氧化损伤保护新靶点。方法　培养细胞系ＡＲＰＥ１９及人原代ＲＰＥ细胞，相差显微镜下观察细胞形态，应用激光共聚焦显微镜免疫荧光法鉴定细胞。不同浓度Ｈ２Ｏ２（０μｍｏｌ·Ｌ－１、１００μｍｏｌ·Ｌ－１、５００μｍｏｌ·Ｌ－１、１ｍｍｏｌ·Ｌ－１）处理细胞２４ｈ后，应用ＲＴ-ＰＣＲ检测ＣｙｃｌｏｐｈｉｌｉｎＤ在细胞内的表达。随后预先在部分细胞中加入３μｍｏｌ·Ｌ－１环孢素Ａ（ｃｙｃｌｏｓｐｏｒｉｎＡ，ＣｓＡ）处理３０ｍｉｎ后再加入不同浓度Ｈ２Ｏ２（８０μｍｏｌ·Ｌ－１、１６０μｍｏｌ·Ｌ－１、３２０μｍｏ·Ｌ－１）２ｈ，即ＣｓＡ＋Ｈ２Ｏ２组，应用乳酸脱氢酶（ｌａｃｔｉｃｄｅｈｙｄｒｏｇｅｎａｓｅ，ＬＤＨ）法检测细胞致死率，并与Ｈ２Ｏ２组的ＬＤＨ释放量相比较。结果　培养的ＡＲＰＥ１９和人原代ＲＰＥ细胞均表达ＲＰＥ细胞特异性抗体ＲＰＥ６５。１００μｍｏｌ·Ｌ－１Ｈ２Ｏ２处理细胞２４ｈ后ＣｙｃｌｏｐｈｉｌｉｎＤ表达量明显升高，当Ｈ２Ｏ２浓度增加到５００μｍｏｌ·Ｌ－１时，ＣｙｃｌｏｐｈｉｌｉｎＤ表达量降低，１ｍｍｏｌ·Ｌ－１Ｈ２Ｏ２处理时，ＣｙｃｌｏｐｈｉｌｉｎＤ表达水平未见增加。在各Ｈ２Ｏ２ 组组间，ＬＤＨ的释放量随着Ｈ２Ｏ２ 浓度的升高而增加；与Ｈ２Ｏ２ 组相比，ＣｓＡ＋Ｈ２Ｏ２组ＬＤＨ的释放水平明显降低（Ｐ＜０．０５）。结论　氧化应激下ＲＰＥ细胞内ＣｙｃｌｏｐｈｉｌｉｎＤ的表达升高，该蛋白表达的增加可能进一步导致氧化应激下细胞的死亡，ＣｓＡ可能成为ＲＰＥ细胞保护的新策略。

Expression of Cyclophilin D in human retinal pigment epithelial cells during oxidative stress

Objective To detect the expression of Cyclophilin D in human retinal pigment epithelial ( RPE ) cells under oxidative stress . and preliminary explore the possible target to protect RPE cells. Methods ARPE 19 cell line was cultured . and human primary RPE cells were observed by phase-contrast micrographs. RPE cells were identified using confocal microscopy. With different concentrations of hydrogen peroxide ( H202) treatment (0 Vmol . 1’1 ,100 Vmol . 1’1 ,500 ymol . 1’1 ,1 mmol . L") for 24 hours.the expression of Cyclophilin D was detected by RT-PCR. The cells were pretreated with 3 ymol . L-l Cyclosporine A ( CsA) for 30 minutes , then different concentrations of H202 ( 80 Vmol . L’l , 160 ymol . L-l , 320 Vmol . L-’) were added to the cells for 2 hours , and the cell death was detected by LDH release between CsA + H202 groups and H202 alone groups. Results Cultured ARPE19 cells and human primary RPE cells were identified by expressing RPE-65 ( a specific marker for these cells) using confocal microscopy. The expression of Cyclophilin D increased significantly after the cells treated with 100 ymol . L-l H202 for 24 hours . decreased when the cells treated with 500 pdmol . L-l H202. No more expression of Cyclophilin D increased in the cells when the concentration of the H202 reached to I mmol . L -’. The release of LDH increased significantly with the increased concentration of H202 in H202 groups. Compared with the H202 alone groups, the release of LDH significantly decreased in the CsA + H2 02 groups( P < 0. 05 ) . Conclusion The expression of Cyclophilin D increase significantly under oxidative stress. which further increase the cell death, suggest that CsA may be a possible target for the protection of RPE cells.