兔骨髓间充质干细胞及人羊膜上皮细胞移植治疗兔角膜缘干细胞缺损的研究  

A comparative study on rabbit bone mesenchymal stem cells and human amniotic epithelial cells transplantation for rabbit limbai stem cell deficiency

作　　者：卢建民[1] 吕秀丽[1] 马翔[1] 

机构地区：[1]大连医科大学附属第一医院眼科,116011

出　　处：《中华实验眼科杂志》2011年第9期786-792,共7页Chinese Journal Of Experimental Ophthalmology

基　　金：国家自然科学基金面上项目（30471859）

摘　　要：背景角膜缘干细胞缺乏可引起致盲性眼病，但传统的治疗方法疗效欠佳。最近的研究表明，骨髓间充质干细胞（BMSCs）和人羊膜上皮细胞（AECs）有分化成多种细胞的能力，但其对角膜缘于细胞缺乏的疗效仍有待研究和评价。目的观察和对比兔BMSCs和人AECs移植治疗兔角膜缘干细胞缺损的治疗效果。方法将18只新西兰白兔采用随机数字表法分为羊膜基质（AS）移植组、兔BMSCs移植组和人AECs移植组，每组6只。将浸有NaOH溶液的滤纸贴附于角膜表面建立碱烧伤角膜缘干细胞缺损的动物模型。抽取兔髂窝处骨髓并收集人胎盘组织分别分离、制备兔BMSCs和人AECs，通过密度梯度离心加贴壁培养法及胰蛋白酶多次分步消化法获取兔BMSCs和人AECs，采用逆转录聚合酶链反应（RT—PCR）法对培养细胞进行鉴定，再接种于人去上皮AS上，按照动物的分组分别将上述材料缝合至动物模型的角膜表面。术后28d，对各组动物的角膜新生血管（CNV）评分以及角膜混浊度评分进行对比，对角膜组织行组织病理学检查并针对角膜上皮细胞特异性标记物细胞角蛋白3（CK3）行免疫组织化学染色。结果第3代兔BMSCs接种于AS载体上12h后贴附生长，第1代人AECs接种于AS载体上48h后呈单层膜状生长，传代细胞经RT—PCR鉴定符合目标细胞的特征。兔BMSCs移植组术后28d，免疫组织化学染色结果显示，兔BMSCs移植组和人AECs移植组的角膜表面细胞CK3均呈阳性表达，而AS组CK3表达阴性。与AS移植组比较，兔BMSCs移植组和人AECs移植组CNV评分以及角膜混浊度评分明显降低，差异均有统计学意义（BMSCs：Z=-2．983，P=0．003；Z=-2．844，P=0．004；AECs：Z=-2．817，P=0．005；Z=-2．041，P=0．041）。组织病理学检查显示，AS组兔角膜组织有较多的炎性细胞生长，胶原纤维排列紊乱。兔BMSCs组术后角膜表层形成了角膜上�Background Limbal stem cell deficiency usually leads to blindness, and traditional therapy is limited. Recent research demonstrated that bone mesenchymal stem ceils （BMSCs） and human amniotic epithelial cells（AECs） could differentiate into many kinds of cells including corneal epithelial cells,but the outcome and effect of these cells on corneal stem cell deficiency are still unclear. Objective This study aimed to observe and compare the effects of rabbit BMSCs and human AECs transplantation for rabbit limbal stem cell deficiency. Methods Eighteen clean New Zealand rabbits were randomly divided into the amniotic stroma（AS） group,rabbit BMSCs group and human AECs group with 6 rabbits for each group. Limbal stem cell deficiency models were established by putting a piece of filter paper that had been soaked in a NaOH solution at the corneal center. Rabbit BMSCs were isolated and purified by density gradient centrifugation combined with the attachment culture method, and human AECs were collected by a sequential trypsin digestion technique, and the third generation rabbit BMSCs and the first generation human AECs were identified with RT-PCR. After that, cells were inoculated onto the denuded AS and grafted to the corneal surface of the experimental animals. Twenty-eight days after cell transplantation, the therapeutic effects were evaluated based on the corneal neovascularization and opacity scores. Corneal histopathological examination and immunohistochemistry were performed to evaluate and compare the effectiveness among AS,rabbit BMSCs and human AECs on corneal stem cell deficiency. The procedure complied with the Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission. Results The third generation of rabbit BMSCs grew well after 12 hours, and the first generation of human AECs formed a membrane-like monolayer after 48 hours of incubation on AS. Immunohistochemistry staining showed that, 28 days after transplantation, the surface cells of

关 键 词：骨髓间充质干细胞 羊膜上皮细胞 角膜缘干细胞 角膜新生血管 干细胞移植 

分 类 号：R77[医药卫生—眼科]