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

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皮样的复层结构，无明显杯状细胞，角膜基质内无新生血管生长，炎性细胞减少，胶原纤维排列更加规则，且兔BMSCs移植组角膜透明度明显优于人AECs移植组，差异有统计学意义（Z=-2．091，P=0．037），而2组间CNV评分的差异无统计学意义（Z=-0．267，P=0．789）。结论移植至兔角膜缘干细胞缺损角膜表面的兔BMSCs和人AECs均能分化为角膜上皮细胞样细胞，可抑制CNV，减轻角膜混浊。在改善角膜透明度方面，兔BMSCs优于人AECs。

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 the cornea were positive for cytokeratin 3 in both the rabbit BMSCs group and human AECs group. Compared with the AS group, the corneal neovascularization and opacity grades were significantly decreased in the rabbit BMSCs group（Z=-2.983,P=0.003;Z=-2.844,P=0.004） and human AECs group（Z=-2.817,P= 0. 005 ; Z = -2. 041, P = 0. 041 ）. Histopathological analysis exhibited that stratified corneal epithelial-like ceils formed on the corneal stroma 28 days after grafting and no signs of goblet cells and neovascularization were found. Less inflammatory cells and regular collagen fiber could be seen in the rabbit BMSCs group and human AECs group. In addition,clinical observation also revealed that the corneas were much clearer in the rabbit BMSCs group than the human AECs group（ Z=-2. 091 ,P=0. 037） ,but the corneal neovascularization score was similar between them（ Z= -0. 267,P=0. 789）. Conclusions Rabbit BMSCs and human AECs can differentiate into corneal epithelial-like cells on the damaged corneal surface and further demonstrate remarkable inhibitory effects on corneal neovascularization and inflammatory cells. The more dominant and prominent effect is the role played by rabbit BMSCs in the improvement of corneal transparency.