三氧化二砷对兔视网膜色素上皮细胞DNA损伤的研究

目的探讨三氧化二砷（As2O3）对视网膜色素上皮（RPE）细胞的损伤作用。方法按照Singh的方法从色素兔眼分离RPE细胞并进行体外培养，培养的细胞用细胞角蛋白进行免疫组织化学鉴定。按照对培养细胞的干预方式不同分为阴性对照组（培养基中加入PBS）、阳性对照组（254nm紫外线照射10min）及实验组。实验组分别在培养基中加入终浓度为50．00、5．00、2．00、1．00、0．50、0．25μmol／L的As2O3作用1h。采用锥虫蓝染色法检测各组细胞的活性。采用彗星实验的方法测定细胞的损伤程度。结果90％以上的培养细胞对细胞角蛋白产生阳性的免疫反应。锥虫蓝染色表明各组细胞的形态和细胞活力均无明显差别。不同组中RPE细胞的尾部DNA含量和尾距的差异均有统计学意义（F=88．548，P=0．000；F=129．704，P=0．000）。与对照组比较，不同浓度的As2O3实验组RPE细胞的尾部DNA含量和尾距明显增加，差异均有统计学意义（P〈0．05），其中50μmol／L组最明显，而0．5μmol／L、0．25μmol／L组与对照组间差异则无统计学意义（P〈0．05）。结论As2O3能够诱导RPE细胞的DNA损伤，损伤程度与As2O3的浓度有关。

Study on DNA damage of retinal pigment epithelium cell induced by arsenic trioxide in rabbit

Background Researches have reported that arsenic trioxide (As_2O_3) is an effective method of treating solid tumor,and its mechanism is to inhibit the cellular proliferation and induce cellular apoptosis.However,the research on DNA damage caused by As_2O_3 is not clear.Objective This study is to investigate the roles of arsenic trioxide (As_2O_3) on DNA damage of retinal pigment epithelial cell in rabbit.Methods Retinal pigment epithelial cells were isolated from pigmented rabbits and cultured in vitro according to the method of Singh NP[2].Cultured cells were identified by cytokeratin.The third or fourth generation of cells were used to this study.50.00,5.00,2.00,1.00,0.50 and 0.25 μmol/L of As_2O_3 were added into medium respectively for 1 hour,and the same volume of PBS was added as negative control group.Cultured cells were exposed to ultraviolet (UV) rays with wave length of 254 nm for 10 minutes as positive control group.The cell vitality was detected by trypan-blue staining.The comet assay was applied to evaluate the DNA damage.SPSS 13.0 software was used for statistical analysis.Results The black granule were seen in cultured RPE cells.Cultured cells showed the positive brown-yellow particles in cytoplasm for cytokeratin with the positive rate over 90%.The morphology of the cells was similar among experimental group,UV irradiation group and PBS group under the inverted microscope.The normal cell activity was exhibited by trypan-blue staining in these three groups.Compared with PBS group,As_2O_3 caused obvious abnormality of DNA of RPE cell in a dose-dependent manner.Both the percentage of tail DNA and tail moment were gradually increased with the enhance of As_2O_3 concentration (F=88.548,P=0.000; F= 129.704,P=0.000).Significant differences also were found between different concentrations of As_2O_3 groups and UV irradiation group compared with PBS group (P＜0.05).As_2O_3 caused the obvious breaking up of DNA strands of RPE cells at the concentration of 50.00 μmol/L,but there was no statistical difference in spontaneous DNA damage of RPE cells between 0.50 and 0.25 μmol/L of As_2O_3 (P＞0.05).Conclusion As_2O_3 has a potential genotoxicity for rabbit retinal pigment epithelial cells in vitro.