眼科研究
眼科研究
안과연구
CHINESE OPHTHALMIC RESEARCH
2010年
2期
130-133
,共4页
视网膜色素上皮%三氧化二砷%DNA损伤
視網膜色素上皮%三氧化二砷%DNA損傷
시망막색소상피%삼양화이신%DNA손상
retinal pigment epithelia%As2O3%arsenic trioxide DNA damage
目的 探讨三氧化二砷(As_2O_3)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As_2O_3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As_2O_3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 As_2O_3 能够诱导RPE细胞的DNA损伤,损伤程度与As_2O_3的浓度有关.
目的 探討三氧化二砷(As_2O_3)對視網膜色素上皮(RPE) 細胞的損傷作用.方法 按照Singh的方法 從色素兔眼分離RPE細胞併進行體外培養,培養的細胞用細胞角蛋白進行免疫組織化學鑒定.按照對培養細胞的榦預方式不同分為陰性對照組(培養基中加入PBS)、暘性對照組(254nm紫外線照射10min)及實驗組.實驗組分彆在培養基中加入終濃度為50.00、5.00、2.00、1.00、0.50、0.25μmol / L的As_2O_3作用1h.採用錐蟲藍染色法檢測各組細胞的活性.採用彗星實驗的方法 測定細胞的損傷程度.結果 90%以上的培養細胞對細胞角蛋白產生暘性的免疫反應.錐蟲藍染色錶明各組細胞的形態和細胞活力均無明顯差彆.不同組中RPE細胞的尾部DNA含量和尾距的差異均有統計學意義(F=88.548,P=0.000;F= 129.704,P=0.000).與對照組比較,不同濃度的As_2O_3實驗組RPE細胞的尾部DNA含量和尾距明顯增加,差異均有統計學意義(P＜0.05),其中50μmol/L組最明顯,而0.5μmol/L、0.25μmol/L組與對照組間差異則無統計學意義(P＜0.05).結論 As_2O_3 能夠誘導RPE細胞的DNA損傷,損傷程度與As_2O_3的濃度有關.
목적 탐토삼양화이신(As_2O_3)대시망막색소상피(RPE) 세포적손상작용.방법 안조Singh적방법 종색소토안분리RPE세포병진행체외배양,배양적세포용세포각단백진행면역조직화학감정.안조대배양세포적간예방식불동분위음성대조조(배양기중가입PBS)、양성대조조(254nm자외선조사10min)급실험조.실험조분별재배양기중가입종농도위50.00、5.00、2.00、1.00、0.50、0.25μmol / L적As_2O_3작용1h.채용추충람염색법검측각조세포적활성.채용혜성실험적방법 측정세포적손상정도.결과 90%이상적배양세포대세포각단백산생양성적면역반응.추충람염색표명각조세포적형태화세포활력균무명현차별.불동조중RPE세포적미부DNA함량화미거적차이균유통계학의의(F=88.548,P=0.000;F= 129.704,P=0.000).여대조조비교,불동농도적As_2O_3실험조RPE세포적미부DNA함량화미거명현증가,차이균유통계학의의(P＜0.05),기중50μmol/L조최명현,이0.5μmol/L、0.25μmol/L조여대조조간차이칙무통계학의의(P＜0.05).결론 As_2O_3 능구유도RPE세포적DNA손상,손상정도여As_2O_3적농도유관.
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.
