丝素蛋白和乳酸-己内酯共聚物纳米纤维支架构建组织工程化角膜上皮的实验研究  

Silk fibroin blended poly (L-lactic acid-co-ε-caprolactone) membrane as a potential substrate for epithelial constructs used in ocular surface bioengineering

作　　者：李纲[1] 钱婷婷[1] 洪佳旭[1] 徐建江[1] 吴继红[1] 崔呈俊[2] 莫秀梅[2] 

机构地区：[1]复旦大学附属眼耳鼻喉科医院眼科,上海200031 [2]东华大学化学化工与生物工程学院生物材料与组织工程实验室,上海201620

出　　处：《中国眼耳鼻喉科杂志》2013年第2期96-99,104,共5页Chinese Journal of Ophthalmology and Otorhinolaryngology

基　　金："十一五"国家科技支撑研究计划(2012BAI08B01);卫生部卫生行业科研专项项目(201002019);国家自然科学基金面上项目(81170817);国家自然科学青年基金项目(81200658);上海市自然科学基金项目(11231200602);上海市科学技术委员会"气象与健康"课题项目(11DZ2260900)

摘　　要：目的评估兔角膜缘上皮细胞在丝素蛋白(SF)和乳酸-己内酯共聚物(PLCL)复合纳米纤维薄膜上的黏附、增殖和分化情况,探讨SF-PLCL复合纳米纤维薄膜作为组织工程角膜上皮支架材料的可行性。方法利用六氟异丙醇(HFIP)作为溶剂,配制浓度为8%的SF和PLCL共混物(质量比25∶75)的纺丝溶液。通过静电纺丝技术制备纳米纤维膜。将体外培养的第一代兔角膜缘上皮细胞种植于SF-PLCL纳米纤维薄膜上进行复合培养。利用扫描电镜观察SF-PLCL纳米纤维薄膜的表面形态及细胞材料复合物的表面形貌;利用免疫荧光染色分析角膜上皮细胞标记物K3的表达情况;通过细胞活力检测观察细胞在材料上的生长分布规律。结果 SF-PLCL纳米纤维薄膜的平均纤维直径为(715±186)nm,材料具有较好的透明度。扫描电镜显示角膜缘上皮细胞在材料上保持正常上皮细胞的铺路石样形态,黏附生长良好,细胞表面布满微绒毛,细胞间紧密联接;免疫荧光染色表明角膜缘上皮细胞在材料上可分化为成熟的角膜上皮细胞。细胞活力检测证实角膜缘上皮细胞在材料上生长良好,均匀分布,形成多层层化结构。结论角膜上皮细胞与SF-PLCL纳米纤维膜的生物相容性良好,SF-PLCL纳米纤维膜是一种潜在、合适的组织工程角膜上皮支架材料。Objective To assess an electrospun silk fibroin （SF） and poly （L-lactic acid-co-s-caprolactone） （PLCL） biomaterial as a subs[rate for corneal epithelial cell adherence, proliferation, and differentiation. To develop a synthetic stromal subs[rate for limbal epithelial cell （LEC） expansion that can serve as a potential subs[rate. Methods Nanofibers were fabricated using 8% SF and PLCL （ weight ratio being 25 ： 75 ） mixed solution dissolved in hexafluoroisopropanol （HFIP） via an electrospinning process. Rabbit limbal epithelial cell on passage one was planted on this membrane. The scaffold and the epithelial constructs were characterized for surface morphology using scanning electron microscopy （SEM）. Immunofluorescent （IF） staining was performed to check the molecular marker expression. Viability staining was used for observing the growth of the rabbit limbal epithelial cells on the membrane. Results The average diameter of SF-PLCL nanofibers was （715 -＋ 186） nm and the nanofiber membrane was high transparent. SEM micrograph showed that the corneal epithelium displayed a polygonal pattern and was firmly anchored to the nanofiher surface. There were abundant microvilli distributed on the surface of the cells, which had wide connection with the adjacent cells. IF staining results revealed that there was no change in the expression profile of LECs grown on nanofibers. The cells were showed to grow well on the nanofibers and form a comparable stratified graft with a 2 to 3 cell layering via viability staining. Conclusions The SF-PLCL membranes could support limbal epithelial cells to proliferate, differentiate, and stratify. This scaffold provides not only a milieu supporting LEC expansion, but also serves as a potential alternative substrate for ocular surface tissue engineering.

关 键 词：丝素蛋白 乳酸-己内酯共聚物 静电纺丝 组织工程 角膜 上皮 

分 类 号：R318.08[医药卫生—生物医学工程]