摘要目的:探讨聚乙二醇水凝胶膜(PHFs)是否能作为角膜上皮细胞(CECs)体外扩增的载体以及其能否用于角膜缘干细胞缺乏症(LSCD)的治疗。方法:利用癸二酰氯、聚氧乙基甘油醚、聚已酸内酯二醇人工合成PHFs，检测其厚度、透光度和机械拉伸性能。取清洁级新西兰大白兔4只，培养原代角膜缘上皮细胞，荧光显微镜下观察培养细胞中角蛋白标志物AE1/AE3和干细胞标志物p63的表达；将细胞分为阴性对照组、阳性对照组和PHFs+CECs组，其中将阴性对照组细胞直接种植在培养皿底，加入普通细胞培养液，将阳性对照组细胞种植在培养皿后加入含有100 μmol/L H 2O 2的普通细胞培养液，将PHF+CECs组细胞种植在铺有PHFs的培养皿中用普通细胞培养液培养，各组培养24 h。采用MTT和TUNEL染色法分别观察3个组细胞增生和细胞凋亡情况。采用随机数字表法将清洁级新西兰大白兔15只分为对照组、PHFs组和PHFs+CECs组，每组5只。其中对照组造模后不给予任何处置；PHFs组将空PHFs膜置于LSCD实验兔角膜表面；PHFs+CECs组为将CECs传代后种植于PHFs上构建组织工程移植片后将移植片置于LSCD兔模型的角膜表面。采用荧光素染色法检测各组实验兔角膜缺损面积大小并评分；采用苏木精－伊红染色法检测各组实验兔角膜上皮组织学特点。 结果:PHFs厚度不超过150 μm，能承受约6 MPa的拉力，在400~700 nm可见光范围内透过率>99%。角膜缘原代培养的大多数细胞AE1/AE3染色和p63染色阳性。MTT实验结果显示，PHFs+CECs组、阴性对照组和阳性对照组 A490值分别为0.59±0.01、0.65±0.07和0.06±0.04，总体比较差异有统计学意义( F＝12.25， P<0.05)，其中PHFs+CECs组和阴性对照组 A490值明显大于阳性对照组，差异有统计学意义(均 P<0.05)。TUNEL检测结果显示，PHFs+CECs组、阴性对照组和阳性对照组TUNEL阳性细胞率总体比较差异有统计学意义( F＝13.45， P<0.05)，其中PHFs+CECs组和阴性对照组TUNEL阳性细胞率明显低于阳性对照组，差异均有统计学意义(均 P<0.05)。荧光素染色结果显示，随着术后时间的延长，各组角膜荧光素钠染色评分均降低，对照组、PHFs组和PHFs+CECs组角膜荧光素钠染色评分依次降低。苏木精－伊红染色结果显示，对照组存在较少形状不规则的角膜上皮细胞，PHFs组一些区域出现单层稀疏的角膜上皮细胞；PHFs+CECs组角膜上皮覆盖范围最大，细胞层数增加到3~5层，形态较规则，排列较紧密。 结论:PHFs韧性强、透光度高且能够体外扩增角膜上皮，适合做角膜上皮移植片，并能促进LSCD兔模型角膜上皮的修复。

abstractsObjective:To investigate whether polyethylene glycol hydrogel films (PHFs) can be used as a carrier for the expansion of corneal epithelial cells (CECs) in vitro and whether PHFs can be used in the treatment of limbal stem cell deficiency (LSCD). Methods:Sebacoyl chloride, dihydroxyl PCL and glycerol ethoxylate were used to synthesize PHFs.The thickness, transmittance and mechanical tensile properties of PHFs were measured.Four clean-grade New Zealand white rabbits were selected to culture primary limbal epithelial cells.The expression of keratin marker AE1/AE3 and stem cell marker p63 in the cultured cells were observed under a fluorescence microscope.The cells were divided into negative control group cultured with common cell culture solution, positive control group cultured with cell culture solution containing 100 μmol/L H 2O 2, and PHFs+ CECs group lined with PHFs cultured with common cell culture solution for 24 hours.The proliferation and apoptosis of cells in the three groups were observed by MTT and TUNEL staining, respectively.Fifteen clean-grade New Zealand white rabbits were divided into control group, PHFs group and PHFs+ CECs group by random number table method, with 5 rabbits in each group.LSCD model was constructed in the three groups.The control group was not given any treatment after modeling.In PHFs group, empty PHFs were placed on the corneal surface of rabbits.In PHFs+ CECs group, tissue-engineered grafts constructed with CECs after passage implanted on PHFs were placed on the corneal surface of rabbits.The corneal defect area of rabbits was detected and scored by fluorescein sodium staining.The histological characteristics of rabbits corneal epithelium was observed by hematoxylin-eosin staining.The use and care of animals complied with Guide for the Care and Use of Laboratory Animals by the U. S.National Research Council.The experimental protocol was approved by the Research and Clinical Trial Ethics Committee of The First Affiliated Hospital of Harbin Medical University (No.2021006). Results:The synthetic PHFs were with a thickness ≤150 μm, a tensile strength about 6 MPa, and a transmittance over than 99% in the range of 400-700 nm.Most of the cells from primary culture of limbal tissue were positive for AE1/AE3 and p63.MTT test results showed that the A490 value of PHFs+ CECs group, negative control group and positive control group was 0.59±0.01, 0.65±0.07 and 0.06±0.04, respectively, showing a statistically significant overall difference ( F=12.25, P<0.05). The A490 values of PHFs+ CECs group and negative control group were significantly higher than that of positive control group, and the differences were statistically significant (both at P<0.05). TUNEL test results showed that there was a significant difference in the TUNEL-positive cell rate among the three groups ( F=13.45, P<0.05), and the rates of TUNEL-positive cells in PHFs+ CECs group and negative control group were significantly lower than that in positive control group (both at P<0.05). Fluorescein sodium staining results showed that with the extension of postoperative period, the corneal fluorescein sodium staining score of the three groups decreased, which decreased successively in control group, PHFs group and PHFs+ CECs group.Hematoxylin-eosin staining showed fewer irregularly shaped corneal epithelial cells in the control group, and sparse single layer of corneal epithelial cells in some areas of the PHFs group.In PHFs+ CECs group, the corneal epithelium coverage was the largest, and the cell layers increased to 3-5, and the cells were with regular morphology and in close arrangement. Conclusions:PHFs have enough toughness, high transmittance and can expand corneal epithelium in vitro.PHFs are suitable for corneal epithelial transplantation and can promote the repair of corneal epithelium in rabbit model of LSCD.