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目的探讨不同的低温冷冻条件对角膜缘上皮细胞生物活性的影响。方法采用不同浓度梯度的二甲基亚砜和不同的降温速率保存兔角膜缘上皮组织;以台盼蓝染色方法计数角膜缘表层上皮细胞的存活率;通过体外细胞培养方法观察低温保存的角膜缘上皮细胞生长情况,并采用若丹明B染色法对原代细胞的增殖能力进行定量测定;将低温保存带有角膜缘的角膜进行器官培养,采用SABC免疫组织化学染色法检测角膜缘上皮细胞的PCNA表达。结果二甲基亚砜浓度梯度对低温保存的角膜缘表层上皮细胞存活率有显著影响,程序降温的效果明显优于非程序降温。体外细胞培养显示,二甲基亚砜浓度梯度和降温方法对角膜缘上皮细胞增殖能力无显著性影响(P>0.05);血清培养组优于无血清培养组(P<0.05,P<0.01)。器官培养1周时,低温保存的角膜缘上皮细胞核中均可见PCNA表达,染色阳性细胞主要在基底细胞层。结论角膜缘干细胞具有较强血清依赖性和低温耐受性,提示超低温保存角膜缘上皮具有可行性。
Objective To investigate the effects of different cryogenic conditions on the biological activity of limbal epithelial cells. Methods The corneal limbal epithelial tissues were preserved in different concentrations of dimethylsulfoxide (DMSO) and different cooling rates. The survival rate of limbal epithelial cells was counted by trypan blue staining. The corneal limbal cryopreservation was observed by in vitro cell culture The growth of epithelial cells and the proliferation of primary cells were quantitatively determined by rhodamine B staining. The cornea with corneal limbal cryopreservation was organ culture. SABC immunohistochemical staining was used to detect the changes of limbal epithelial cells PCNA expression. Results The DMSO gradient had a significant effect on the survival rate of limbal epithelial cells preserved at low temperature. The cooling effect of the program was obviously better than that of non-program cooling. Cell culture in vitro showed that DMSO concentration gradient and cooling method had no significant effect on the proliferation of limbal epithelial cells (P> 0.05); serum culture group was better than serum-free culture group (P <0.05, P <0.01) . One week after organ culture, PCNA expression was observed in the limbal epithelial cell nucleus preserved at low temperature, and the staining positive cells were mainly in the basal cell layer. Conclusion The corneal limbal stem cells have strong serum-dependent and low-temperature tolerance, suggesting that cryopreservation of limbal epithelial cells is feasible.