人胚胎视网膜短、中、长期保存的实验研究

Short-, mid- and long-term preservation of human fetal retina

目的观察全层人胚胎视网膜的短期、中期及长期保存后的活性和结构。方法12-24周全层人胚胎视网膜22例分为88片，明胶包被后分别行Ames液、中期保存液（DX角膜保存液）、-80℃、程序深低温4种方法保存不同时间后，锥虫蓝染色观察视网膜神经上皮层活细胞百分率，光学显微镜和透射电子显微镜观察视网膜组织结构。结果人胚胎视网膜在新鲜取材时活细胞占（94．79±2．85）％；短期保存在Ames液4h内活细胞百分率大于80必，中期保存在DX液1～2d时活细胞百分率大于77％，均与新鲜取材时活细胞百分率无显著差异。-80℃保存7d时活细胞百分率为（65．83±5．06）％，1个月时降至（57．54±16．18）％；深低温长期保存1个月时活细胞百分率为（69．46±9．31）％。全层人胚胎视网膜保存在Ames液2h时光学显微镜和电子显微镜检查均未见明显异常；在DX液中2d、-80℃7d和深低温1个月时光学显微镜检查见其层次清楚，但细胞间隙加大，组织排列疏松。结论全层人胚胎视网膜在Ames液和DX角膜保存液中可于一定时间内保持较好的活性和组织结构。

Objective To observe the configuration and viability of full thickness human fetal retina after short-, mid and long-term preservation. Methods Twenty two full thickness human fetal retinae of gestational age of 12 24 weeks were coated by glutin and cut into 88 pieces, and then preserved in Ames＇ solution, DX solution, - 80℃ refrigerator or under cryopreservation condition. The cell viability of retinal neuroepithelial layer was determined by trypan blue staining, retinal configuration was determined by light microscope and electromicroscope. Results The viability of neuroepithelial layer was （94.79±2.85） % in fresh fetal retina, 〉80% in Ames＇ solution within 4 hours, and 〉77% in DX solution within 2 days. There was no significant difference between those solution preservations and the fresh fetal. In -80℃ refrigerator, the viability was （65.83±5.06）% after 7 days, and then dropped to （57.54± 16.18）% at the end of the first month. Under the cryopreservation condition, the viability was （69.46±9.31）% at the end of first month. Light and transmission electron microscopy had not deteced any abnormals in the full thickness human fetal retina preserved in Ames＇ solution within 2 hours, but showed clear retinal layers with bigger intercellular space after preserved in DX solution for 2 days, in -80℃ refrigerator for 7 days and under cryopreservation condition for 1 month. Conclusion Ames＇ solution and DX solution can preserve good viability and configuration of full thickness human fetal retina in a certain time period.