培养的兔角膜缘上皮细胞深低温保存后的生物学活性研究

Study on biologic viability of cultural rabbit's limbal corneal epithelial cells after cryopreservation

目的探讨深低温保存角膜缘上皮细胞的可行性和效果。方法将兔角膜缘组织培养出的细胞,分别用两组冷冻保护液冻存,0.5、1、2个月后分批取出。将未冷冻的细胞和不同冻存时间、不同冻存保护液的冷冻复苏后细胞传代培养,采用免疫组化、电镜鉴定培养后细胞的性质。通过倒置显微镜、电镜、MTT比色法来比较传代培养的角膜缘上皮细胞深低温保存前后的形态结构和生物学活性。结果AE1和传代细胞鼠抗兔增殖细胞核抗原(PCNA)单克隆抗体染色呈阳性反应,AE5单克隆抗体染色偶见阳性反应;冻存后的传代细胞贴壁、生长均滞后,而且电镜检查均有不同程度细胞水肿,再培养7d后形态结构均恢复正常;MTT法检测复苏当天冻存后的细胞比未冻存的细胞增殖活性低,差异显著(P<0.05,但培养5d后无显著差异(P>0.05),甘油组和DMSO组间有显著性差异(P<0.05),不同冻存时间的各冻存组测得值无显著差异(P>0.05)。结论冻存的角膜缘上皮细胞传代培养仍保持上皮细胞特性并含有干细胞;DMSO保护液比甘油液低温保存效果好;角膜缘上皮细胞经阶段降温后深低温保存简单可行,选择适当的冷冻保护剂冻存后的细胞再培养,其细胞活性和结构与原代相似。

Objective To search for the feasibility and the effect that cultural limbal corneal epithelial cells were cryopreserved. Methods Small specimens of limbal epithelial cells were excised from the limbal regions in rabbits, and were cultivated. Some monolayer cells were digested into suspending liquid and were cryopreserved in two groups of cryoprotectant solutions respectively. Cryopreserved ceils were frozen in liquid nitrogen and were thawed after half a month, a month and two month differently. The cultivated limbal epithelial cells were identified by immunohistochemical and electron microscope. The structure and activity of frozen and non-frozen cells were compared by inverted microscope, electron microscope and MTT colorimetry. Results Subeulturing cells were positive staining for monoclonal antibody cytokeratin AE1 and anti-proliferating cell nuclear antigen（ anti-PCNA）, were rarely positive for monoclonal antibody cytokeratin AE5. The adherence and growth of cryopreserved cells delayed compared with that of the fresh cells. Electron microscope revealed that cryopreserved cells appeared different extent dropsy and the recovery of the cell ultrastructure were in 7 days. MTr colorimetry proved that the proliferation of frozen cells were lower than that of non-frozen cells after thawing （ P 〈 0.05 ） and after 5 days of subeulturing（ P 〉 0.05 ）. There were no significant differences among the detective values of different frozen time groups （ P 〉 0.05 ）, whereas the difference was statistically significant between that of the dimethyl sulfoxide group and the glycerol group （ P 〈 0.05 ）. Conclusion Limbal corneal epithelial cells frozen kept the characteristics of epithelial cells. After these thawed cells were continued cultivating, there were a lot of stem cells that had ability in proliferation in subeulturing cells. The cryopreservative effect of dimethyl sulfoxide was better than that of glycerol. The approach of stage freezing was simple and feasible in order to cryopreserve cultural limbal corneal epithelial cells. These cells which were frozen by the reasonable cryoprotectant had similar stucture and function after subculturing.