Abstract: Objective　To evaluate the feasibility of using peripheral blood mononuclear cells (PBMCs) as an alternative cell source to corneal limbal stem cells envisaging corneal epithelial regeneration. Methods　The experiment was conducted from January 2020 to January 2021, and 30 healthy New Zealand white rabbits aged from 5 to 8 months were selected from the physiological laboratory of Jinan University. Culture and identification of PBMCs in vitro: the rabbit PBMCs were isolated by density gradient centrifugation, the morphology of PBMCs was observed under inverted phase contrast microscope, and the CD14, a surface marker of monocytes, was detected by flow cytometry. PBMCs were cultured in the microenvironment of fresh limbal stem cells and corneal epithelial cells for one week. The positive rate of cytokeratin, a specific marker on cell surface, was detected by flow cytometry before and after induction. Analysis of variance was used to compare the positive expression rate index of cell surface keratin before and after induction. t test was used for the measurement data. Results　The positive rate of cytokeratin of PBMCs was 1.64% before induction by flow cytometry; the positive rate of cytokeratin of PBMCs was 11.30% after induction by flow cytometry. Before induction, there was no statistically significant difference in the positive expression rate of cytokeratin between the experimental group and the control group (P>0.05); after 7 days of induction, the positive expression rate of cytokeratin in the experimental group was (10.298±0.996)%, which was significantly higher than that in the control group [(1.786±0.237)%] (P<0.05). The positive expression rate of cytokeratin in the experimental group was (1.308±0.376)% before induction and (10.298±0.996)% after 7 days of induction, with a statistically significant difference (P<0.05). There was no statistically significant difference in the positive expression rate of cytokeratin in the control group before induction and after 7 days of induction (P>0.05). Conclusion　PBMCs as an alternative autologous cell source for limbal stem cells is envisaged as an effective therapeutic strategy for corneal surface reconstruction.

Key words: Peripheral blood mononuclear cells, Induction of differentiation, Corneal epithelial cells, Co-culture model

摘要： 目的　探讨外周血单核细胞作为角膜缘干细胞替代角膜上皮细胞再生的潜能。方法　实验时间为2020年1月至2021年1月，实验动物为来自暨南大学生理实验室5～8月龄健康新西兰大白兔30只；外周血单核细胞体外培养及鉴定：采用密度梯度离心法分离兔外周血单核细胞，在倒置相差显微镜下观察单核细胞形态，细胞分选技术检测单核细胞表面标志物CD14。将收集的单核迁移细胞置于新鲜角膜缘和角膜上皮组织块的微环境中共培养1周，流式细胞仪检测诱导前与诱导后细胞表面特异标志物细胞角蛋白的阳性率，诱导前与诱导后细胞表面角蛋白阳性表达率指数的组间比较采用重复测量设计的方差分析。计量资料采用t检验。结果　流式细胞术检测诱导前单核迁移细胞特异标志物细胞角蛋白阳性率为1.64%，诱导后单核迁移细胞特异标志物细胞角蛋白阳性率为11.30%。诱导前，实验组细胞角蛋白阳性表达率与对照组比较，差异无统计学意义（P>0.05）；诱导7 d后，实验组细胞角蛋白阳性表达率为（10.298±0.996）%，与对照组（1.786±0.237）%比较，差异有统计学意义（P<0.05）；实验组诱导前细胞角蛋白阳性表达率为（1.308±0.376）%，与诱导7 d后（10.298±0.996）%比较，差异有统计学意义（P<0.05）；对照组诱导前细胞角蛋白阳性表达率与诱导7 d后比较，差异无统计学意义（P>0.05）。结论　外周血单核细胞作为角膜缘干细胞的自体替代干细胞来源，被认为是角膜上皮重建的有效治疗策略。

关键词: 外周血单核细胞, 诱导分化, 角膜上皮细胞, 共培养系统