原代培养角膜缘干细胞与ΔNp63蛋白表达的年龄相关性研究  

Age-related study on primary culture of limbal stem cells and the expression of ΔNp63 protein

作　　者：闫实[1] 马蕾[1] 

机构地区：[1]山西医科大学第二医院眼科,太原030001

出　　处：《眼科研究》2009年第3期193-196,共4页Chinese Ophthalmic Research

基　　金：山西省卫生厅科技攻关计划项目资助(200706)

摘　　要：目的建立兔角膜缘干细胞(LSCs)原代培养体系,了解年龄的增长对兔LSCs的ΔNp63蛋白表达的影响。方法选取3月龄、1.5年龄及3年龄新西兰白兔各10只,用含10%胎牛血清的DMEM/F12培养液对LSCs的细胞悬液进行体外原代培养。应用逆转录聚合酶链反应(RT-PCR)技术和间接免疫荧光法分别检测培养细胞中ΔNp63蛋白的表达量。结果接种3d后大部分细胞贴壁,6d左右形成良好的细胞单层,细胞呈圆形、椭圆形或多边形,类似角膜上皮细胞。年龄越大,ΔNp63的表达量越低。不同年龄组之间ΔNp63表达量的差异有统计学意义(P<0.05)。结论含10%胎牛血清的DMEM/F12培养液能作为LSCs培养的基础液;ΔNp63蛋白的表达具有明显的年龄相关性,随年龄的增长ΔNp63表达量的下调可能是LSCs老化的分子机制之一。Objective It have been well-known that the lack of limbal stem cells（LSCs） results in the abnormality of ocular surface.The study on the in vitro culture of LSCs for the treatment of ocular surface diseases has been concerned.The purpose of this study was to establish a method for cultivating rabbit LSCs primarily and investigate the influence of aging on ΔNp63 protein expression in LSCs.MethodsNew Zealand white rabbits with the age 3 months,1.5 years and 3 years were selected in this study and 10 rabbits for each group.The lameller corneal limble tissue was isolated and cultured in medium containing 10% fetal bovine serum（FBS） using mass cultivation method.LSCs were collected and identified by ΔNp63 immunofluorescence stain.RT-PCR technique and indirect immunofluorescence were performed to assess the change of expression of ΔNp63 protein with aging.ResultsCultured LSCs adhered and grew within 3 days and formed a confluent layer 6 days later with the shape similar to LSCs and presented the green fluorescence in the cell nucleus for ΔNp63.The fluorescence intensity of ΔNp63 protein expression was 127±24,96±22 and 48±15 in 3-month-old,1.5-year-old and 3-year-old rabbits respectively,showing a statistically significant difference among these three groups（F=20.535,P〈0.05）.The gray scale value of ΔNp63 mRNA expression was 0.7633±0.0136,0.6340±0.0134 and 0.3655±0.0116 in 3-month-old,1.5-year-old and 3-year-old rabbits respectively,indicating a obvious difference in an age-dependent manner（F=1451.222,P〈0.05）.Conclusion10%FBS culture medium can be used as the basic medium for LSCs culture.The expression of ΔNp63 protein in LSCs is associated with age,which suggest that ΔNp63 protein may play an important role in the aging process of LSCs.

关 键 词：角膜缘 干细胞 衰老 ΔNp63蛋白 

分 类 号：R772[医药卫生—眼科]