以猪角膜板层为载体诱导人骨髓间充质干细胞治疗兔角膜损伤的初步研究

发布时间：2018-11-10 20:19

【摘要】： 目的探讨以猪角膜前板层基质为载体诱导人骨髓间充质干细胞(Mesenchymal Stem Cells, MSCs)治疗兔角膜损伤的可能性。方法用全骨髓贴壁法分离纯化人MSCs,将细胞接种于含10%胎牛血清(FBS)的DMEM培养基中培养扩增,传代,流式细胞仪检测免疫表型及诱导成脂、成骨分化,采用油红O及硝酸银染色鉴定。自动板层角膜刀钻取直径约7.5 mm,厚160μm的去上皮猪角膜基质片。28只新西兰白兔随机分为2组,制作角膜广泛损伤模型。实验组取第3代MSCs接种于去上皮和全周角膜板层缘的猪角膜基质上,培养4天后移植到广泛损伤的兔角膜上,对照组单纯移植去上皮猪角膜基质。术后2、4、8周观察实验眼角膜透明度和新生血管并评价分析。术后2、4、8周,取各实验眼行组织学检查,观察移植的MSCs及猪角膜基质的存活、转归及移植局部的反应。免疫组织化学、免疫荧光染色检测移植后角膜上皮细胞标志物细胞角蛋白12(Cytokeratin 12, CK12)的表达。结果获得的细胞流式细胞仪检测：CD29阳性率为95.97%,CD44为96.49%,CD90为92.79%,CD105为94.66%,CD34为0.59%,CD45为0.36%。符合MCSs的免疫表型,并可以诱导成脂及成骨分化。接种到去上皮猪角膜基质后贴附、生长迅速。术后植片在植床上存活良好,未见明显排斥反应,实验组角膜较对照组明显透明,新生血管少。实验组角膜免疫组织化学及免疫荧光均检测出CK12阳性细胞。结论人骨髓MSC可以在体外成功进行原代和传代培养,体外培养的人骨髓MSC有很强的增殖能力。种植MSCs的猪角膜基质移植到损伤兔角膜后可以存活并减少兔角膜新生血管、增加透明度,本实验条件下MSCs可以分化为角膜上皮样细胞,可能具有构建组织工程角膜的潜能。
[Abstract]:Objective to investigate the possibility of rabbit corneal injury induced by human bone marrow mesenchymal stem cell (Mesenchymal Stem Cells, MSCs) with porcine anterior lamellar matrix. Methods Human MSCs, was isolated and purified by whole bone marrow adherent method. The cells were cultured and amplified in DMEM medium containing 10% fetal bovine serum (FBS). The immunophenotype, adipose induction and osteogenic differentiation were detected by flow cytometry. Oil red O and silver nitrate staining were used. The epitheliized porcine corneal stroma with the diameter of 7.5 mm, and 160 渭 m was obtained by automatic lamellar keratectomy. 28 New Zealand white rabbits were randomly divided into two groups to make extensive corneal injury model. The third generation of MSCs was inoculated on the porcine corneal stroma which was epithelially removed and the entire limbus cornea was cultured for 4 days, then transplanted to the widely damaged rabbit cornea, while the control group was only transplanted to the epithelial porcine corneal stroma. Corneal transparency and neovascularization were observed and evaluated 8 weeks after operation. Four weeks after operation, the experimental eyes were examined by histological examination to observe the survival, prognosis and local reaction of transplanted MSCs and porcine corneal stroma. Immunohistochemistry and immunofluorescence staining were used to detect the expression of Cytokeratin 12 (CK12) in corneal epithelial cells after transplantation. Results the positive rate of CD29 was 95.97 and the CD44 was 96.499.The CD90 was 92.79 and the CD105 was 94.666.The CD34 was 0.59and the CD45 was 0.360.The results showed that the positive rate of CD29 was 95.97% and the CD44 was 96.49% and the CD90 was 92.79%. It conforms to the immunophenotype of MCSs and can induce adipogenic and osteogenic differentiation. After inoculation to epithelial porcine corneal stroma, it grows rapidly. After operation, the grafts survived well in the bed, and there was no obvious rejection. The cornea in the experimental group was more transparent than that in the control group, and the neovascularization was less than that in the control group. CK12 positive cells were detected by immunohistochemistry and immunofluorescence in the experimental group. Conclusion Human bone marrow MSC can be successfully cultured in vitro. Human bone marrow MSC has strong proliferative ability. The porcine corneal stroma implanted with MSCs can survive and reduce the corneal neovascularization and increase the transparency. Under this condition MSCs can differentiate into corneal epithelioid cells which may have the potential to construct tissue engineered cornea.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R772.2

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