原子力显微镜在平板离心法筛选的人角膜缘上皮细胞研究中的应用  

Atomic force microscopy analysis of human corneal epithelial stem cells fractionated by rapid novel centrifugation isolation technique

作　　者：张伟[1] 杨为中[1] 金敏[1] 高宗银[1] 陈晓燕[2] 许艳丽[2] 张玉平[2] 

机构地区：[1]广州市第一人民医院眼科,广东省广州市510180 [2]广州市第一人民医院血液实验室,广东省广州市510180

出　　处：《眼科新进展》2012年第3期211-214,共4页Recent Advances in Ophthalmology

基　　金：广州市卫生局医药卫生科技项目基金资助(编号:20121-A011011);广州市卫生局医药卫生科技项目(编号:201102A212025)~~

摘　　要：目的用原子力显微镜研究平板离心法筛选出的三群角膜缘上皮细胞,确定细胞的类型。方法用人角膜缘组织块培养法原代培养出角膜缘上皮细胞,收获细胞后将细胞置于未包被的六孔板上1400r.min-1和1800r.min-1进行离心分群。ATC1为1400r.min-1离心后贴壁的细胞,ATC2为1800r.min-1离心后贴壁的细胞,NAC为1800r.min-1离心后漂浮而不能贴壁的细胞。用BrdU标记延迟实验和原子力显微镜来分析这三群细胞。结果在三群细胞中ATC1几乎全被BrdU标记延迟,占分选前总体细胞的10.14%±1.21%。原子力显微镜显示ATC1、ATC2和NAC的细胞形态和细胞膜表面有显著的差异。整体细胞粗糙度在ATC1、ATC2和NAC中分别为(20.91±1.42)nm、(80.37±3.43)nm和(102.94±4.52)nm,核部位膜的粗糙度分别为(33.79±1.66)nm、(13.12±0.79)nm和(8.48±0.64)nm。结论原子力显微镜是一种非常好的区分和观察三群细胞的方法。BrdU标记延迟实验和原子力显微镜分析参数表明ATC1、ATC2和NAC可能分别为角膜缘干细胞、短暂扩充细胞和终末分化细胞。Objective To investigate the use of atomic force microscopy（AFM） to image the three groups of human corneal epithelial cells fractionated by rapid novel centrifugation isolation technique.Methods Human corneal epithelial cells harvested from primary cultures of limbal rings were allowed to centrifugation in uncoated dishes with 1400 r·min-1 and 1800 r·min-1,sequentially.The adherent cells with 1400 r·min-1（ATC1）,adherent cells with 1800 r·min-1（ATC2） and non-adherent cells with 1800 r·min-1（NAC） were investigated for BrdU-label retention and contact mode AFM.Results Among un-fractionated cells and three selected populations,the ATC1,accounting for about（10.14±1.21）% of whole population,was enriched in BrdU label-retaining cells.There were dramatic overall shape and surface membrane differences among ATC1,ATC2 and NAC.The whole cell roughness measurements were（20.91±1.42）nm,（80.37±3.43）nm and（102.94±4.52）nm in ATC1,ATC2 and NAC,respectively.But the mero-nucleus roughness measurements were（33.79±1.66）nm,（13.12±0.79）nm and（8.48±0.64）nm in ATC1,ATC2 and NAC,respectively.Conclusion AFM was a good tool for detecting and distinguishing the cells among the three groups.BrdU-label retention and the AFM parameters showed that ATC1,ATC2 and NAC may be progenitor corneal epithelial cells,transit amplifying cells and terminal differentiation cells,respectively.

关 键 词：原子力显微镜 平板离心法 人角膜缘干细胞 

分 类 号：R977.6[医药卫生—药品]