新型角膜上皮细胞载体促进角膜碱烧伤修复的作用研究

发布时间：2018-08-15 15:56

【摘要】：目的评价羧甲基壳聚糖(Carboxymethyl chitosan,CMCTS)共混膜片的理学及生物学性能,并研究其在角膜碱烧伤修复中的作用。方法将CMCTS与明胶(Gelatin,Gel)和透明质酸(Hyaluronic acid,HA)按照20∶1∶2的比例制备CMCTS共混膜片,使用全波长酶标仪于400~800 nm波长下检测该膜片的透光性;将膜片植入大鼠皮下及肌肉组织,组织切片HE染色评价其组织相容性和炎症反应;通过大鼠肝脏和肾脏的体重质量比评估膜片在体内的降解毒性。将兔原代角膜上皮细胞接种于CMCTS共混膜片上构建角膜上皮细胞移植体,CFSE染色和扫描电镜观察细胞在膜片上的活性和生长状态;将该细胞移植体用于新西兰大白兔角膜碱烧伤眼表的治疗,通过外眼相观察、裂隙灯观察和组织免疫荧光染色与空白对照组、模型组及没有细胞移植的膜片组相比较,评价其修复效果。结果本研究制备的CMCTS共混膜片在400~800 nm波长下透光率均高于80%;大鼠肌肉及皮下植入膜片2 w后,组织切片HE染色可见周围组织炎症反应基本消失;膜片在皮下及肌肉组织中能够稳定降解,在皮下组织中降解时间超过8 w,而在肌肉组织中4 w完成降解;膜片在降解过程中对肝、肾均无明显毒性;CFSE染色和扫描电镜观察兔原代角膜上皮细胞能够在膜片上贴附生长并连接成片;将其移植到角膜碱烧伤的兔眼表,发现其在术后30 d内基本完成创面修复,而单纯膜片组的修复能力较弱,模型组仅靠角膜自身修复作用不能进行角膜碱烧伤的完全修复。结论本实验中制备的CMCTS共混膜片具有良好的物理及生物学性能,对兔角膜碱烧伤具有明显的治疗效果,为今后角膜组织工程方法治疗角膜碱烧伤提供了可靠的实验依据。
[Abstract]:Objective to evaluate the physical and biological properties of Carboxymethyl chitosan (CMCTS) blend film and to study its role in corneal alkali burn repair. Methods CMCTS, gelatinine and hyaluronic acid (HA) were used to prepare the CMCTS blend film at 20:1:2. The transmittance of the film was detected by using a full-wavelength enzyme label at 400nm, and the film was implanted into the subcutaneous and muscle tissues of rats. The histocompatibility and inflammatory response were evaluated by HE staining and the biodegradation toxicity of the membrane was evaluated by the body mass ratio of liver and kidney in rats. Rabbit primary corneal epithelial cells were inoculated on CMCTS blend membrane to construct corneal epithelial cell graft. The activity and growth state of corneal epithelial cells on the membrane were observed by scanning electron microscope (SEM). The cell grafts were used in the treatment of alkali burn of cornea in New Zealand white rabbits. The results were compared with those of control group, model group and membrane group without cell transplantation by external eye phase observation, slit lamp observation and tissue immunofluorescence staining. The effect of restoration was evaluated. Results the transmittance of the CMCTS blend films prepared in this study was higher than that of 80% at the wavelength of 400 ~ 800nm, and the inflammatory reaction of the surrounding tissues was basically disappeared by HE staining in the tissue sections of the rats after 2 weeks of muscle and subcutaneous implantation. The membrane could degrade steadily in subcutaneous and muscle tissues, and the degradation time in subcutaneous tissue was more than 8 ws, but in muscle tissue for 4 weeks. CFSE staining and scanning electron microscopy were used to observe the primary corneal epithelial cells attached to the membrane and connected to the rabbit corneal surface, which were transplanted to the surface of the cornea after alkali burn, and the wound repair was basically completed within 30 days after operation. However, the repair ability of the simple membrane group was weak, and the model group could not completely repair the corneal alkali burn only by the corneal self-repair. Conclusion the CMCTS blend film prepared in this experiment has good physical and biological properties and has obvious therapeutic effect on alkali burn of cornea in rabbits. It provides a reliable experimental basis for the treatment of alkali burn of cornea by corneal tissue engineering in the future.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R779.1

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