眼科研究
眼科研究
안과연구
CHINESE OPHTHALMIC RESEARCH
2009年
12期
1064-1067
,共4页
角膜缘上皮细胞%荧光激活细胞分离技术%侧群细胞%ABCG2%K3/K12
角膜緣上皮細胞%熒光激活細胞分離技術%側群細胞%ABCG2%K3/K12
각막연상피세포%형광격활세포분리기술%측군세포%ABCG2%K3/K12
limbal epithelium cells%fluorescence-activated cell sorting%side-population cells%ABCG2%K3/K12
目的 探讨荧光激活细胞分离技术(FACS)在体外培养的兔角膜缘上皮细胞生物学特性研究中的应用.方法 收集新西兰白兔的角膜缘组织进行组织块培养.采用FACS分离出体外培养的兔角膜缘上皮细胞中的侧群(SP)细胞和非SP细胞.用2%锥虫蓝染色法对分离出的SP细胞进行克隆形成率(CFE)检测以评估SP细胞的活性,用免疫组织化学染色检测 K3/12和ABCG2蛋白在SP细胞和非SP细胞中的表达.结果 SP细胞在体外培养的兔角膜缘上皮细胞中占(0.22±0.09)%,分离出的SP细胞的CFE为(5.52±0.45)%,而非SP细胞为(0.78±0.73)%,二者差异有统计学意义(t＝2.17,P<0.01);大部分SP细胞呈K3/K12抗原阴性,ABCG2免疫标记呈阳性;而非SP细胞呈K3/K12抗原阳性,ABCG2免疫标记呈阴性.结论 FACS可以应用于体外培养的兔角膜缘上皮细胞的SP细胞分离,分离出的SP细胞具有较强的增生能力.
目的 探討熒光激活細胞分離技術(FACS)在體外培養的兔角膜緣上皮細胞生物學特性研究中的應用.方法 收集新西蘭白兔的角膜緣組織進行組織塊培養.採用FACS分離齣體外培養的兔角膜緣上皮細胞中的側群(SP)細胞和非SP細胞.用2%錐蟲藍染色法對分離齣的SP細胞進行剋隆形成率(CFE)檢測以評估SP細胞的活性,用免疫組織化學染色檢測 K3/12和ABCG2蛋白在SP細胞和非SP細胞中的錶達.結果 SP細胞在體外培養的兔角膜緣上皮細胞中佔(0.22±0.09)%,分離齣的SP細胞的CFE為(5.52±0.45)%,而非SP細胞為(0.78±0.73)%,二者差異有統計學意義(t＝2.17,P<0.01);大部分SP細胞呈K3/K12抗原陰性,ABCG2免疫標記呈暘性;而非SP細胞呈K3/K12抗原暘性,ABCG2免疫標記呈陰性.結論 FACS可以應用于體外培養的兔角膜緣上皮細胞的SP細胞分離,分離齣的SP細胞具有較彊的增生能力.
목적 탐토형광격활세포분리기술(FACS)재체외배양적토각막연상피세포생물학특성연구중적응용.방법 수집신서란백토적각막연조직진행조직괴배양.채용FACS분리출체외배양적토각막연상피세포중적측군(SP)세포화비SP세포.용2%추충람염색법대분리출적SP세포진행극륭형성솔(CFE)검측이평고SP세포적활성,용면역조직화학염색검측 K3/12화ABCG2단백재SP세포화비SP세포중적표체.결과 SP세포재체외배양적토각막연상피세포중점(0.22±0.09)%,분리출적SP세포적CFE위(5.52±0.45)%,이비SP세포위(0.78±0.73)%,이자차이유통계학의의(t＝2.17,P<0.01);대부분SP세포정K3/K12항원음성,ABCG2면역표기정양성;이비SP세포정K3/K12항원양성,ABCG2면역표기정음성.결론 FACS가이응용우체외배양적토각막연상피세포적SP세포분리,분리출적SP세포구유교강적증생능력.
Objective The fluorescence-activated cell sorting (FACS) technique is a method for the identification and isolation of different cell populations.At present,the special surface marker for limbal stem cells has been not found yet.This study aimed to investigate the application of FACS technique in the research of rabbit limbal stem cells.MethodsCorneal limbal tissue was obtained from New Zealand white rabbits and cultured using the explant culture method in SHEM.Side population cells (SP cells) and non-SP cells were sorted from cultured rabbit limbal epithelium cells by FACS at a excitation wavelength 350 nm,and acquistion length 450 nm (blue light) and 675 nm (red light).The SP cells and non-SP cells were identified by detecting the expression of ABCG2 and K3/K12.The colony-forming efficiency of SP cells and non-SP cells were evaluated by the observation of cellular vitality with trypan blue staining.The percentage of colony formation was calculated as the colony number in various group/200×100%.ResultsIn 48-72 hours after primary culture,limbal epithelial cells migrated from the cultured tissue mass to form the mambrane-like structure and achieved 70%-80% confluence.The cells showed round,polygon and flattened shape.The proportion of SP cells in cultured limbal epithelial cells was 0.22%±0.09% with a colony-forming efficiency of 5.52±0.45% in SP cells and 0.78%±0.73% in non-SP cells,with a statistically significant difference between the two populations (t＝2.17,P＜0.01).After verapamil,an inhibitor of the expression of the ABCG2 protein,was added into the medium,the proportion of SP cells in the cultured limbal epithelial cells declined to 0.04%±0.006%.The SP cells presented a positive immunoresponse for ABCG2 and absence of immunoresponse for K3/K12,but a contradictory staining result was found in non-SP cells.ConclusionFACS can be applied in the research of limbal stem cells.
