组织工程角膜上皮治疗兔角膜缘干细胞缺乏症的研究  

Transplantation of bioengineered corneal epithelium for the treatment of total limbal stem cell deficiency in rabbit

作　　者：蔺琪[1] 吕岚[1] 韩斌 金涛[1] 

机构地区：[1]首都医科大学北京同仁眼科中心,100730 [2]北京以岭生物工程有限公司

出　　处：《中华眼科杂志》2006年第8期679-685,共7页Chinese Journal of Ophthalmology

基　　金：国家高技术研究发展计划(863计划)基金资助项目(2003AA205070-04)

摘　　要：目的评价以纤维凝胶膜为载体,体外培养自体角膜缘干细胞移植术治疗角膜缘干细胞缺乏症的临床效果。方法制作兔眼角膜缘干细胞完全缺乏模型,取对侧眼角膜缘组织进行干细胞培养,并以纤维凝胶膜为载体制成组织工程角膜上皮。将实验动物随机分为4组,其中Ⅰ-Ⅲ组为实验组,行组织工程角膜上皮移植术,Ⅱ组移植术后观察3个月,Ⅱ组移植术后观察1个月,Ⅲ组移植术后观察2周。Ⅳ组为对照组,移植不含干细胞的凝胶膜,术后观察3个月。以角膜上皮染色、角膜混浊及新生血管3项指标进行临床疗效评定,通过病理检查评估术后不同时期角膜上皮修复情况,印迹细胞学检查移植前后角膜上皮的细胞表型,免疫组织化学染色观察角膜上皮特异性角蛋白K3、MUC5AC及转录因子p63在移植角膜上皮的表达。结果实验组角膜上皮逐渐愈合,透明度提高,新生血管减退或消失;对照组角膜呈持续混浊,上皮缺损迁延不愈,最终大量新生血管长入,上皮结膜化。临床指标评分比较,差异有统计学意义(P=0．021)。角膜上皮印迹细胞学检查显示对照组为PAS(+)的结膜细胞表型,而实验组上皮细胞PAS(-)。组织病理学显示实验组为正常角膜上皮结构,对照组为结膜化生的上皮,可见血管和杯状细胞。免疫组织化学显示实验组角膜上皮表达角膜上皮特异性角蛋白K3(AE5),不表达结膜特异性MUCSAC,在上皮基底部表达转录因子p63。对照组上皮持续表达MUC5AC。结论组织工程角膜上皮移植术是一种有效的治疗角膜缘干细胞缺乏症的方法,可重建眼表,修复角膜上皮的损伤,抑制角膜上皮结膜化和新生血管。纤维凝胶膜为一种新型的组织工程材料,吸收快,透明度高,具有良好的组织相容性,是一种较理想的移植载体材料。Objective To study the results of transplantation of cultured autologous limbal stem cells using fibrin gel membrane as substrate for the treatment of limbal stem cell deficiency in rabbits. Methods An ocular surface defect was created in the right eyes of all rabbits by a lamellar keratectomy extending 1 mm outside the limbus. The stem cells were isolated from a small limbal biopsy specimen from the left eyes and cultured. Cells were grown over fibrin gel membrane to construct bioengineered corneal epithelium. After cytologic verification of total limbal stem cell deficiency, rabbits were divided into 4 groups at random. The fibrovascular pannus of each cornea was removed. Groups Ⅰ -Ⅲ （n = 24 ） underwent transplantation of limbus stem cells cultured on fibrin gel membrane and were observed for 3 months （ Group Ⅰ ）, 1 month （ Group Ⅱ ） and 2 weeks （ Group Ⅲ ） , whereas group Ⅳ （n = 8） received only the fibrin gel membrane. Clinical outcome was graded by corneal integrity, opacity and neovascularization. HE staining, impression cytology and immunofluorescence staining with antibodies against keratin-3, MUCSAC and nuclear p63 were performed to assess the phenotype of corneal epithelium. Results The cornea in the study groups （group Ⅰ-Ⅲ ） recovered smoothly, covered with transparent epithelium and without neovascularization. In the control group （group Ⅳ ）, cornea was turbid and irregular, with neovascularization （ P =0.021 ）. Corneal impression cytology indicated PAS（ - ）in the study groups, while PAS（ ＋ ）in the control group. HE staining of the cornea in the study groups showed normal corneal phenotypes; whereas the epithelium cells in the control group showed conjunctival phenotypes with vessels and goblet cells. Immunostaining of study groups showed keratin-3 positive and MUCSAC negative corneal epithelium, and 1363 was expressed in the basal cell of corneal epithelium. Cells in the control group expressed MUC5AC abundantly. Conclusions Tissue bioengine

关 键 词：组织工程 上皮 角膜 角膜疾病 干细胞移植 

分 类 号：R772.21[医药卫生—眼科]