摘要
背景目前角膜移植仍是治疗严重角膜疾病的主流方法，但角膜供体的不足使角膜移植疗法的应用受到限制。组织工程角膜上皮为角膜移植研究提供了新的途径，其中组织工程角膜的支架研究也是研究热点之一。细菌纤维素膜已作为生物材料广泛用于医药领域，其在组织工程角膜的支架研究方面的应用值得关注。目的以细菌纤维素膜作为支架，研究体外培养的兔角膜上皮细胞在细菌纤维素膜上生长情况，评估细菌纤维素膜的生物相容性。方法收集1月龄的新西兰白兔角膜上皮组织，采用组织块培养法对角膜上皮细胞进行培养和传代，采用免疫荧光技术检测细胞中CK-3的表达对培养的细胞进行鉴定。将第2代兔角膜上皮细胞消化后分别接种于细菌纤维素膜和培养皿，光学显微镜下观察和比较两组细胞的生长情况。于接种后第3天角膜上皮细胞在细菌纤维素膜上贴壁达到融合时加入10ml含5μl钙黄绿素-AM＋20μl溴乙非啶同二聚体的无菌PBS（LIVE／DEAD细胞活性毒性检测），评估2种培养法细胞的活性率。细菌纤维素膜上培养的细胞固定后，扫描电子显微镜下检查角膜上皮细胞的表面超微结构。结果兔角膜上皮细胞原代培养后1周左右细胞生长良好，呈铺路石样排列并融合，免疫荧光检测显示培养的细胞CK-3表达阳性。接种于细菌纤维素膜上的细胞生长状态良好，呈圆形或椭圆形，呈铺路石样排列，LIVE／DEAD染色后活细胞细胞核呈绿色荧光，细胞存活率为100％。扫描电子显微镜下细菌纤维素膜上培养的兔角膜上皮细胞呈椭圆形，可见膜表面丰富的页状突起、丝状伪足及细胞间连接，并可观察到处于分裂相的细胞。上皮细胞通过丝状伪足紧密贴附于细菌纤维素膜。结论兔角膜上皮细胞在细菌纤维素膜上生长状态良好，细菌纤维素膜有较好的生物相容性，可作为构建组织工程角膜上皮的一种新型生物学材料。
Background Corneal transplantation is a primary method for the treatment of serious corneal diseases, but its application is limited because of the shortage of corneal donor. The study on tissue engineering corneal epithelium provides a new approach to corneal transplantation, and the biological scaffold materials for tissue engineering corneal epithelium is an issue of increasing concern. Bacterial cellulose membrane has been used in medical field,but its application in tissue engineering corneal epithelium deserves more researching. Objective This study was to evaluate the biocompatibility of bacterial cellulose membrane as a biological scaffold of tissue engineering corneal epithelium. Methods Corneal epithelium was isolated from 1 month-old New Zealand White rabbit. Corneal epithelial ceils were cultured using explant method and identified by detecting the CK-3 expression using immunofluorescence technique. The second generation cells were inoculated on bacterial cellulose membrane and culture plate, respectively, and the growth status of the cells were examined and compared under the optical microscope. The cell activity/toxicity test was performed by LIVE/DEAD cell staining kit at the third day after inoculation to evaluate the survival rate. The ultrastructure of the cell surface was examined under the scanning electron microscope. The study was performed in accordance with the ARVO Statement. Results Rabbit corneal epithelial cells grew well 1 week after primarily cultured with a cobblestone-like appearance and positive response for CK3 antibody. The cells on the bacterial cellulose membrane presented a round shape and regular arrangement and showed the green fluorescence for LIVE/DEAD test,with the survival rate 100%. Abundant leafy protrusion, microvilli and intercellular junction were seen under the scanning electron microscope. In addition, mitosis phase of cells and many filopodia between the cells and bacterial cellulose membrane were also exhibited. Conclusions Rabbit corneal epithelial cells can grow well in bacterial cellulose membrane. Bacterial cellulose membrane has good biocompatibility,indicating that bacterial cellulose membrane can be used as new biological material for tissue engineering corneal epithelium.
出处
《中华实验眼科杂志》
CAS
CSCD
北大核心
2016年第2期121-124,共4页
Chinese Journal Of Experimental Ophthalmology
基金
国家自然科学基金青年基金项目（81200663）
关键词
角膜上皮/细胞学
组织工程
组织支架/微生物
生物相容材料
细胞培养
兔
细菌纤维素
Epithelium, corneal/cytology
Tissue engineering
Tissue scaffolds/microbiology
Biocompatiblematerials
Ceils, cultured
Rabbits
Bacterial cellulose
