人角膜缘干细胞的体外培养及鉴定  

Primary culture and biological characterization of human limbus stem cell in vitro

作　　者：魏彤心[1] 李冰[1] 史静华[1] 

机构地区：[1]山西省眼科医院角膜病科,太原030002

出　　处：《眼科研究》2010年第5期432-435,共4页Chinese Ophthalmic Research

基　　金：山西省科学技术厅科技公关项目(031061-1)

摘　　要：目的探讨人自体角膜缘干细胞(LSCs)体外培养及鉴定的方法。方法将供体新鲜人角膜缘组织在制备的含20%胎牛血清DMEM/F12培养基的羊膜载体上应用组织块培养法进行体外培养,对培养获得的LSCs用苏木精-伊红染色在倒置显微镜下进行形态学观察,用免疫荧光技术鉴定培养的LSCs细胞。结果组织块静置2h后贴壁良好,3～4d细胞从组织块边缘萌出,7～10d细胞出现生长高峰,12～14d形成良好的细胞单层。细胞形态呈多边形、圆形、卵圆形和梭形。第14天时行间接免疫荧光染色,可见大量细胞表达p63,呈细胞质的绿色荧光;少量细胞表达AE5,呈细胞质的绿色荧光和细胞核的红色荧光。结论组织块培养法体外培养的人LSCs具有与正常人LSCs相一致的生物学特性。Background Limbus stem cells （LSCs） are the source of cornea epithelium,and cuhured LSCs have the same biological characterization as original ones. They can be transplanted to surface of damaged eye to resume the function of limbus. Objectives The present study was to explore the methods of human LSCs cuhure and identify in vitro. Methods The amnion dressing was obtained from fresh human body placenta during the abdominal delivery and put the prepared amnion in 3.5 cm cuhure dish. Human lamellar limbus tissue was obtained from the eyes of donor and cuhured in amnion culture dish with DMEM/F12 containing 20% fetal bovine serum by expland cuhure method. The cells struclure was observed by H＆E staining under the inverted microscope. Cuhured cells were identified by immunofluorescence staining on the 14th day. The Human Research Ethics Committee of this hospital approved this trail. The written informed consent was obtained from all the pregnant woman prior to the medicine protocol. Results The limbus tissue mass adhered to dish well 2 hours after culture. LSCs erupted from the tissue mass on the 3rd-4th day and monolayer formed in the 12nd-14th day. The cellular structure was intact and close intercellular junction and cell-amnion junction were seen under the inverted microscope. Majority of cells were positive green fluorescence for p63 and only a tew cells were stained into green fluorescence in cytoplasm and red fluorescence in nucleus for AE5. Conclusion Human LSCs can be cuhured successfully on amnion membrane, and cuhured cells have the same bionomies feature as normal human LSCs in vivo.

关 键 词：角膜缘干细胞 羊膜 体外培养 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]