低温冷冻对角膜缘干细胞生物学特性的影响

目的 评价低温冷冻保存对体外培养兔角膜缘干细胞生物学特性的影响.设计实验研究.研究对象新西兰白兔.方法 新西兰白兔20只（40眼）,制备2 mm周边角膜和2 mm球结膜的环形浅层角膜缘植片,待形成细胞单层后,将培养的细胞低温冻存1个月,然后复苏培养.将未冻存的细胞和冻存复苏后的细胞采用免疫细胞荧光染色、流式细胞仪检测,鉴定培养细胞的生物学特性,通过倒置显微镜、MTT比色法比较传代培养的角膜缘干细胞低温保存前后的形态结构和特性.主要指标角膜缘干细胞活性及生物学特性.结果 显微镜下观察冻存复苏后的细胞,与未冻存细胞相比,细胞状态差,贴壁时间晚,核浆比变小,形态不规则;免疫荧光细胞染色检测ABCG2单克隆抗体阳性率未冻存细胞为81.7%,低温冻存细胞降至46.9%,差异有统计学意义（P〈0.05）;流式细胞仪检测低温冻存细胞与未冻存细胞角膜缘干细胞占总细胞的比例分别为0.90%和2.43%,差异有统计学意义（P〈0.05）;MTT法检测低温冻存的细胞比未冻存细胞增殖活性降低,差异有统计学意义（P〈0.05）.结论 低温冻存后角膜缘干细胞的活性和生物学特性均受到严重影响,其冻存方法和复苏技术有待进一步改进.

Effects of cryopreservation on biological characteristics of cultivated corneal limbal stem cell

Objective To explore the effects of cryopreservation on biological characteristics of cultured limbal stem cells. Design Experimental study. Participants Twenty New Zealand white rabbits. Methods Twenty New Zealand white rabbits （40 eyes） were preparated of peripheral cornea with 2 mm and 2 mm circular shallow conjunctival limbal graft. Rabbit limbal stem cells （Rb-LSCs） used for cell-suspention culture were isolated form New Zealand white rabbit limbal tissues. The limbal stem cells, having formated monolayer cultured limbal stem cells, were cryopreservated one month, then thawing. By immunofluoreseence staining, flow cytometry, inverted microscope, MTF assay, we compared the subculture of limbal stem cells cryopreservation before and after the structure and properties. Main Outcome Measurements Limbal stem cell activity and biological characteristics. Results Compared with freshing cells, the cells after cryopreservation in morphology were poor adherence late, small nuclear cytoplasm ratio, irregular shape. Im- munofluorescence staining to detect the low-temperature frozen cells ABCG2 monoclonal antibody 81.7% positive rate decreased to 46.9%, the difference was statistically significant （P 〈0.05）. Limbal stem cell percentage of total cells with and without frozen cells were 0.90% and 2.43% by flow cytometry, the statistical analysis showed significant difference （P 〈0.05）; MTY assay showed that the prolif- eration activity of frozen cell decreased significantly （P 〈0.05） than those without. Conclusions Cryopreservation seriously affects the activity and biological characteristics of limbal stem cell. The technology of the cryopreservation and recovery need to be further improved.