改良兔角膜细胞冻存与复苏方法的实验研究  

Improving ways to cryopreservation and resuscitation of keratocytes of rabbit in vitro

作　　者：葛红岩[1] 殷秀丽[1] 付安安[1] 张红[1] 宋甄[1] 李志坚[1] 刘平[1] 

机构地区：[1]哈尔滨医科大学第一临床医学院眼科医院,黑龙江省哈尔滨市150001

出　　处：《眼科新进展》2007年第9期670-674,共5页Recent Advances in Ophthalmology

摘　　要：目的建立一种改良的兔角膜细胞冻存和复苏的方法。方法兔角膜细胞消化培养。取第2代细胞分别进行传统和改良方法冻存,于冻存后的第2周、3个月、6个月、1a分别行传统和改良的复苏方法。用MTT法测细胞生长曲线,应用三因素分析统计方法评定不同冻存方法、不同复苏方法、不同复苏时间三因素单独和(或)组合对细胞复苏率的影响。结果兔角膜细胞体外生长良好。培养细胞Vim单克隆抗体阳性。细胞的复苏率均受不同冻存方法和复苏方法单、双因素影响,不受复苏时间及与冻存和复苏方法单、双因素及三因素的影响。冻存复苏后生长曲线良好。结论改良后的兔角膜细胞冻存和复苏方法可使细胞保持最佳生物学特性,细胞复苏率高,适用于体外实验研究。Objective To establish a modified method of cryopreservating and resuscitating keratocytes of rabbit in vitro. Methods Keratocytes of rabbit was digested with trypsin in vitro and passaged one time, then were identified and saved by conventional and improved cryopreservation methods. The frozen cells were resuscitated after 2 weeks, 3 months, 6 months and one year by two resuscitation methods. The cell growth curve of frozen cells was measured by MTT colorimetry and three factors analysis were performed. Results Keratocytes exhibited good growth during planting culture. The cultured cells stained positively for monoclonal antibody vimentin. The recovered rates of the frozen clumps were affected by cryopreservation methods and resuscitation methods, respectively and by the different combinations. No influence was shown by resuscitation time, the others different combinations and three factors combination. The cell growth curve of frozen cells measured by MTT colorimetry was similar to that of nonfrozen cells. Conclusion We can get the better biologic phenotypes of keratocytes and the highest liability by the modified methods of cryopreservation and resuscitation. Rabbit keratocytes are suitable for experiment in vitro with the improved method.

关 键 词：角膜细胞 冻存 复苏 兔 

分 类 号：R772[医药卫生—眼科]