角膜基质细胞诱导分化的脂肪间充质干细胞羊膜片移植治疗兔角膜碱烧伤的疗效及其机制  

Efficacy and mechanism of rabbit corneal stromal cell-induced adipose-derived mesenchymal stem cell amnion transplantation for corneal alkali burn

作　　者：李颖[1] 杨磊[2] 宋艳萍[2] 丁琴[2] 陈中山[2] 陈晓[1] 

机构地区：[1]南方医科大学附属武汉临床学院,广州510515 [2]广州军区武汉总医院眼科,武汉430070

出　　处：《中华实验眼科杂志》2015年第6期500-506,共7页Chinese Journal Of Experimental Ophthalmology

基　　金：湖北省自然科学基金项目（2011CDB025）

摘　　要：背景角膜化学烧伤是致盲眼病之一，先前的各种疗法在临床上的应用均受到一定的限制，而脂肪间充质干细胞（ADSCs）移植疗法受到密切关注。目的观察诱导分化的ADSCs羊膜片移植治疗兔角膜碱烧伤的疗效及其机制。方法无菌条件下剪取兔角膜，用悬浮基质片法分离并培养兔角膜基质细胞（CSCs）。取兔腹股沟脂肪组织，以酶消化法（质量分数0．25％胰蛋白酶）分离及培养兔ADSCs，并用流式细胞术鉴定ADSCs表面抗原的表达。将CSCs与ADSCs共培养，采用免疫荧光技术和逆转录PCR（RT-PCR）鉴定CSCs诱导后ADSCs向角膜基质样细胞分化的表达，分别将诱导或未诱导的ADSCs种植于羊膜上制备成细胞羊膜片。选择60只新西兰白兔，右眼用浸有质量分数1％NaOH的滤纸贴敷于角膜中央50S制备兔角膜碱烧伤模型，按随机数字表法将模型眼随机分为诱导ADSCs＋羊膜移植组、未诱导ADSCs＋羊膜移植组、单纯羊膜移植组及模型组。分别于术后1周、2周和1个月在裂隙灯显微镜下观察各组兔模型眼角膜的混浊程度和新生血管形成情况，并对角膜炎症进行评分，采用ELISA法检测术后1个月角膜组织匀浆中CD45、叮干扰素（IFN-γ）、白细胞介素．10（IL-10）蛋白及房水中血管内皮生长因子（VEGF）蛋白的质量浓度。结果从脂肪组织分离培养的ADSCs表面抗原CDl05、CD29、CD44的阳性表达率分别为90．23％、88．56％、98．88％；分离培养的CSCs波形蛋白呈阳性表达；CSCs与ADSCs共培养后多数细胞双标记染色阳性。苏木精一伊红染色显示，ADSCs种植于羊膜后生长良好。术后1个月，模型组角膜混浊呈瓷白色，新生血管面积最大，而诱导ADSCs＋羊膜移植组角膜透明，新生血管最少。术后1个月，诱导ADSCs＋羊膜移植组、未诱导ADSCs＋羊膜移植组、单纯羊膜移植组和模型组兔角膜混浊程度评分分别为1．65±Background Corneal chemical burn is one of blinding eye diseases. Previous therapies forcorneal chemical burn is limited to certain extent. However,transplantation of adipose-derived mcsenchymal stem cells （ADSCs） for corneal diseases is drawing more and more attention. Objective This study was to observe the effect of rabbit ADSCs transplantation for ocular alkali burns and explore its mechanism. Methods Rabbit corneal stromal ceils （CSCs） were isolated and cultured by suspended matrix method,and rabbit ADSCs were obtained and digested from inguinal fat tissue with enzyme digestion method （0.25% trypsin） and identified by flow cytometry. CSCs cocultured with ADSCs,and CSCs-induced ADSCs were identified by double-label of with immunofluorescence and reverse transcription PCR （RT-PCR）. Then induced or uninduced ADSCs were inoculated on amniotic membrane to prepared ADSCs-amnion patch. Corneal alkali burn models were established in the right eyes of 60 New Zealand rabbits by placing a filter paper with 1% NaOH solution at the central cornea for 50 seconds. The models were randomized into the induced ADSCs ＋ amnion implanted group, the uninduced ADSCs ＋ amnion implanting group, amnion implanted group and model group. Corneal opacification and neovascular area were examined and corneal inflammation was graded by slit lamp microscope 1 week,2 weeks and 1 month after surgery. The contents of CD45, interferon-γ（IFN-γ） and interleukin-lO （IL-IO） in corneal homogenate as well as vascular endothelial growth factor （VEGF） in aqueous humor were detected by ELISA assay. The use and care of experiment animals followed the Statement of ARVO. Results ADSCs showed the positive responses for CD105 ,CD29,CD44 with the positive rate 90. 23% ,88.56% and 98.88% , respectively. CSCs was positively reactive for vimentin. The double-label staining was positive after cocuhure of CSCs with ADSCs. Hematoxylin-eosin stain exhibited that ADSCs grew well on the amnion. Corneal porcelain opacity and a lot of ne

关 键 词：脂肪间充质干细胞 移植 羊膜 眼碱烧伤 分化 角膜基质细胞 

分 类 号：R779.13[医药卫生—眼科]