氧化石墨烯对人胚胎干细胞诱导的视网膜色素上皮细胞活性的影响  

Effect of graphene oxide on cell viability of human embryonic stem cells derived retinal pigmental epithelial cells

作　　者：曾玉晓[1] 吴畏[2] 徐海伟[2] 阴正勤[2] 史春梦[1] 

机构地区：[1]第三军医大学军事预防医学院全军复合伤研究所创伤、烧伤与复合伤国家重点实验室,重庆400038 [2]第三军医大学西南医院全军眼科中心视觉损伤与再生修复重庆市重点实验室,重庆400038

出　　处：《第三军医大学学报》2016年第12期1410-1415,共6页Journal of Third Military Medical University

基　　金：国家重点基础研究发展计划(973计划,2013CB967002)~~

摘　　要：目的探索氧化石墨烯(graphene oxide,GO)对人胚胎干细胞(human embryonic stem cell,h ESC)诱导的视网膜色素上皮细胞(retinal pigment epithelial,RPE)活性的影响。方法 h ESCs通过自发分化法诱导为RPE细胞,通过免疫荧光法和透射电镜对其进行鉴定后,将h ESC-RPE细胞与浓度分别为0、12.5、25、50和100μg/m L的GO共培养24 h,采用CCK-8试剂盒检测其细胞活力,与浓度为50μg/m L的GO共培养24 h,流式细胞仪检测细胞凋亡和细胞内反应性活性氧(reactive oxygen species,ROS)水平。结果 h ESC分化的RPE细胞,呈典型的铺路石样形态,细胞间连接紧密,胞浆富含色素颗粒;免疫荧光检测表达RPE细胞早晚期及紧密连接的标记,并具有吞噬感光外节能力;电镜观察到顶端的微绒毛,细胞呈单层;GO经原子力显微镜检测,说明制备的氧化石墨烯溶液以单层为主;经紫外-可见吸收以及拉曼光谱测试表明石墨经过强氧化之后,其有序的结构遭到破坏,产生了许多缺陷和出现了大量的无序结构;CCK-8细胞活性检测结果显示各组均无统计学差异;细胞凋亡和氧化应激实验中与空白组之间差异没有统计学意义。结论 GO对于h ESC-RPE细胞生物相容性好,提示其有望作为RPE细胞的纳米载体。Objective To determine the effect of graphene oxide（ GO） on the cell viability of human embryonic stem cells（ h ESCs）-derived retinal pigmental epithelial（ RPE） cells. Methods RPE cells were derived from h ESCs via natural differentiation. After identification by immunofluorescence assay and transmission electron microscope（ TEM）,h ESCs-derived RPE cells were co-cultured with 0,12. 5,25,50 and 100 μg / m L GO for 24 h. Cell viability was tested by CCK-8 kit. Reactive oxygen species（ ROS） was tested with DCFH-DA probe. Results h ESCs-derived RPE cells showed a classical paving-stone shaped morphology with tight connections between the cells and abundant pigment particles in the cytoplasma.Immunofluorescence assay showed that RPE cells expressed the early- and late-stage markers as well as tight junction markers. Besides,RPE cells showed the ability to phagocytize the outer segment of photoreceptors.RPE cells were arranged in a single layer,and the microvilli were observed by TEM. CCK-8 assay showed no significant difference among different groups. ROS had no statistically significant difference among the treatment groups and the control group. With an atomic force microscope,GO solution was proved to be monolayer. The results of ultraviolet-visible spectrophotometry and Raman spectrometry showed that after strong oxidation of graphene,the original ordered structure had been broken,with production of large amount of defect and unordered structure. Conclusion GO has considerate biocompatibility with h ESCs-derived RPE cells,which implies that GO is the potential nano-carrier for RPE cells.

关 键 词：人胚胎干细胞 RPE细胞 氧化石墨烯 细胞活性 

分 类 号：R322.91[医药卫生—人体解剖和组织胚胎学] R392-33[医药卫生—基础医学]