深低温保存兔角膜缘干细胞的体外培养  

In Vitro Culture of Cryopreserving Rabbit Limbal Stem Cells

作　　者：易敬林[1] 钟文贤[1] 杨海军[1] 钟琳娜[2] 

机构地区：[1]江西省人民医院眼科,江西南昌330006 [2]丰城市人民医院眼科,江西丰城331100

出　　处：《江西医学院学报》2005年第6期8-11,F0002,共5页Acta Academiae Medicinae Jiangxi

基　　金：[江西省科委社会发展攻关资助项目]

摘　　要：目的探寻深低温保存兔角膜缘干细胞的体外培养方法。方法将2 mm宽的兔角巩膜组织通过程序降温仪降温后置于-196℃液氮中深低温保存。1年后复温,应用消化培养法分别对冻存及新鲜的兔角膜缘干细胞进行体外培养,观察并记录细胞生长情况,HE染色观察培养细胞的形态特征。结果经过深低温保存的兔角膜缘干细胞可以在体外被成功培养并传代,其在原代培养48h后开始贴壁生长,5 d后生长较旺盛,但可见部分破裂和死亡细胞,约12 d基本形成单层;传代培养时次日贴壁,7 d形成单层。新鲜兔角膜缘干细胞原代培养时细胞形态相似,但破碎死亡细胞少见,且于7 d左右即基本形成单层,传代时两者无明显差别。结论深低温保存的角膜缘干细胞能被成功地进行体外培养,培养后细胞具有与培养的新鲜角膜缘干细胞基本相似的特性,为深低温保存的角膜缘干细胞培养后移植治疗临床眼表疾病提供了实验依据,并为眼库技术的发挥开辟了新的应用途径。Objective To explore the culture methods of cryopreserving rabbit limbal stem cells in vitro. Methods Rabbit corneoscleral tissues in width of 2 mm were cryogenerated by programcontrolled cryogenertor,then they were cryopreserved in--196℃ liquid nitrogen. After one year, they were rewarmed and cultured by enzymatic digestion culture method compared with fresh rab- bit limbal stem cells. The growth characteristics of the cultured cells were observed and recorded, and the morphologic features of the cells were analyzed by the staining of hematoxylin and eosin. Results The cryopreserved limbal stem cells of rabbit in profound hypothermua could be successfully cultured in vitro. The limbal stem cells of primary culture began to adhere and grow after 48 hours,and the cells grew prosperously at the 5 th day, but some ruptured and dead cells could be seen. The cells formed a confluent layer in 12 days. In subculture,the cells adhered to the wall of culture dish next day and formed a monolayer in 7 days. The primary culture cells of fresh limbal stem cells were similar with that of cryopreserved cells in morphology, but the ruptured and dead cells were not common and the cells formed a monolayer in 7 days. The two kind of limbal stem cells were not different in subculture. Conclusion Cryopreserved limbal stem cells in profound hypothermua can be successfully cultured in vitro by enzymatic digestion culture method, and the characteristics of them are similiar with that of cultured cells from fresh limbal stem cells of rabbit. This study supports the treatment of clinical ocular surface disorders by cultured limbal stem cells cryopreserved in profound hypothermua and develops a new pathway of application of eye bank.

关 键 词：角膜缘 干细胞 培养 深低温保存 

分 类 号：Q813.11[生物学—生物工程]