牛磺酸对经单细胞凝胶电泳技术检测人视网膜色素上皮氧化损伤的保护特征

Protection characteristics of taurine on oxidative damage of human retinal pigment epithelial cells detected by single cell gel electrophoresis

目的:观察过氧化氢对人视网膜色素上皮细胞的氧化作用,以及牛磺酸对人视网膜色素上皮细胞的抗氧化作用。方法:实验于2005-01/09在哈尔滨医科大学公共卫生学院中心实验室完成。①人眼视网膜色素上皮细胞来源:实验眼来自20～35岁男性意外死亡后12h内捐给黑龙江省眼库作为角膜移植的供体眼5眼,家属签署知情同意书。取8～10代人眼视网膜色素上皮细胞用于实验。②过氧化氢损伤实验:将培养的细胞按随机数字表法分为5组:过氧化氢各浓度组分别加入终浓度为10,25,50,100μmol/L的过氧化氢,阴性对照组只加入等量的磷酸盐缓冲液。③牛磺酸实验:将培养的细胞按随机数字表法分为5组:阴性对照组用不含血清的DMEM-F12培养液培养,牛磺酸各浓度组分别用含有终浓度为300和600μmol/L的牛磺酸培养液进行培养。将上述每个浓度组的细胞悬液分成2份,向其中1份加入终浓度为25μmol/L的过氧化氢,另一份加入等量的磷酸盐缓冲液。④指标检测:15min后,用单细胞凝胶电泳法检测人视网膜色素上皮细胞的DNA损伤的程度,以拖尾率(彗星细胞数/总细胞数)和拖尾细胞DNA迁移距离[400倍显微镜下测量25个拖尾细胞的DNA迁移长度,整个核DNA直径和迁移DNA(即彗星尾)长度,其差值为DNA迁移距离]的变化表示。⑤统计学分析:用四格表χ2检验(确切概论法)比较组间彗星细胞拖尾率;用t检验进行组间DNA迁移距离比较。结果:①过氧化氢损伤实验结果:阴性对照组DNA荧光图像为红色圆球形,过氧化氢各组出现彗星样,随着过氧化氢浓度(10,25,50,100μmol/L)的增加,人视网膜色素上皮细胞拖尾率和DNA迁移距离均明显高于或长于阴性对照组[拖尾率:4.6%,26.0%,43.3%,86.6%,96.0%;DNA迁移距离:(4.13±0.52),(10.54±2.03),(20.59±4.42),(39.56±10.28),(51.54±15.93)μm,P<0.01]。②牛磺酸实验结果:300和600μmol/L牛磺酸组视网膜色素上皮细胞拖尾率和拖尾细胞DNA迁移距离与阴性对照组相近(P>0.05)。300和600μmol/L牛磺酸+25μmol/L的过氧化氢组均明显低于或短于25μmol/L过氧化氢组[拖尾率:22.6%,20.0%,43.3%;DNA迁移距离:(17.46±7.13),(13.56±2.52),(20.59±4.42)μm,P<0.05],且牛磺酸浓度越高,差异越大。结论:10μmol/L过氧化氢就能对人视网膜色素上皮细胞直接造成DNA断裂损伤,该作用呈剂量依赖性。适量的牛磺酸(300和600μmol/L)对人视网膜色素上皮细胞DNA的氧化断裂损伤起保护作用。

AIM： To observe the oxidation of hydrogen peroxide （H202） on human retinal pigment epithelial （hRPE） ceils, and the protective effect of taurine on oxidative injuries of hRPE cells. METHODS： The experiment Was conducted in the Central Laboratory of Public Health College of Harbin Medical University from January to September in 2005.①The sources of hRPE cells： The experimental eyes were from the male donors aged 20-35 years supplied to the Eye Bank of Heilongjiang Province within 12 hours of accidental deaths, with the informed signatures and consents of their relatives. The 8^th-10^th passage hRPE cells were adopted in this experiment. ②H2O2 damage experiment： The cultured hRPE ceils were treated with different concentrations of H2O2 （10, 25, 50 and 100 μmol/L）, while control group was treated with equal phosphate buffer solution （PBS） only.③ Taurine experiment： The cultured hRPE ceils were pretreated with taurine of 300 and 600 μmol/L, whereas control group was pretreated with DMEM-F12 culture medium without serum. Then the cell suspension of former two groups were divided into 2 subgroups respectively： 25 p, mol/L H2O2 were added into one group and the other was added with equal PBS.④Detections of index： Fifteen minutes later, single cell gel electrophoresis （SCGE） was used to assay the DNA damages of hRPE ceils. The tailing ratio （number of comet cells/total number of cells） and variation of DNA migration length of tailing ceils [DNA migration lengths of 25 tailing cells were measured under microscope （x400）, and the devalue of whole nucleus DNA diameter and migrated DNA （comet tail） length was defined as the DNA migration length] were monitored for the assessment of DNA damage. ⑤Statistical analysis： The tailing ratio of comet cells between groups were compared with fourfold table x^2 test, while the t test was used to compare the DNA migration length between groups. RESULTS： ①The experimental results of H2O2-induced damage： H2O2 caused significant DNA migration of hRPE ceils. Cells with DNA damage appeared fluorescent comets with tails of DNA fragmentation, and the tailing ratio of hRPE ceils and DNA migration length of tailing ceils were obviously higher or longer, with the increases of H2O2 concentrations [Tailing ratio： 4.6%, 26.0%, 43.3%, 86.6%, 96.0%; DNA migration length： （4.13±0.52）, （10.54±2.03）, （20.59±4.42）, （39.56±10.28）, （51.54±15.93） μm, P 〈 0.01]. In contrast, cells with undamaged DNA appeared round spots.② Taurine experiment： There was similarity in tailing ratio of hRPE cells and DNA migration length of tailing cells between control group and taurine groups of 300 and 600 μmol/L （P 〉 0.05）. But the levels of DNA damage of hRPE cells were significantly decreased in taurine groups of 300 and 600 μmol/L＋25 μmol/L H2O2 than in 25 μmol/L H2O2 group [Tailing ratio： 22.6%, 20.0%, 43.3%; DNA migration length： （17.46±7.13）, （13.56±2.52）, （20.59±4.42） μm, P 〈 0.05]. In addition, the differences were more significant, with the higher concentration of taurine. CONCLUSION： H2O2 of 10 μmol/L can induce the DNA fragmentation damage in hRPE cells directly, whereas taurine of suitable doses （300 and 600 μmol/L） can reduce the oxidative damage of hRPE cells DNA fragmentation damage, Both of the effects are dose-dependent.