以猪角膜脱细胞基质为载体培养人脐静脉内皮细胞构建实验性角膜后板层

 目的： 探讨以异种后弹力膜/基质为载体诱导人脐静脉内皮细胞向角膜内皮细胞分化的可行性及移植术后在活体的生理功能。方法：体外分离培养脐静脉内皮细胞作为诱导移植的种子细胞；取当地质检合格的猪眼球以直径6.2 mm、厚100 μm、冷冻脱水进行角膜深板层载体的制备；接种经CM-DiI荧光标记的第2~3代人脐静脉内皮细胞于载体的后弹力层面，体外培养7~10 d行细胞形态、密度、组织学和扫描电镜观察，待细胞与后弹力层面融合形成单层后，用于兔角膜移植。受眼：正常健康新西兰大白兔24只（24眼），实验组（人脐静脉内皮细胞移植组）12眼，对照组（单纯猪角膜深板层移植组）12眼，全角膜范围去除术眼内皮细胞，实施移植手术。结果：人脐静脉内皮细胞在猪后弹力膜/基质载体上贴附生长，形成紧密连接的单层，形态近似六角形，呈铺路石状分布，具有正常兔角膜内皮细胞的超微结构。术后8周实验组角膜基本透明，周边角膜略有水肿。对照组单纯猪角膜深板层移植后，移植角膜明显水肿、混浊。结论：以异种角膜后弹力膜/基质为载体培养的人脐静脉内皮细胞，行异种异体移植后，能够在活体上成活，并能维持角膜透明，具有正常角膜内皮细胞的生物学功能。深板层角膜内皮移植术是体外培养血管内皮细胞移植的一种有效术式。

Decellularized porcine corneal posterior lamellae as carrier matrix for cultivating human umbilical vein endothelial cells in vitro

AIM：To investigate the feasibility of corneal posterior lamellar reconstruction with human umbilical vein endothelial cells (HUVECs) and porcine cornea acellular matrix in vitro, and to observe the physiological function of the transplantation in vivo. METHODS：HUVECs were isolated, cultured, and labeled with fluorescent dye CM-DiI. Porcine corneas were treated with 100% glycerinum, cut to a thinner structure step by step, and dried on the super-clean bench. Transmission electron microscope were used to observe the histological changes of the porcine cornea acellular matrix. Labeled HUVECs were seeded onto the porcine cornea acellular matrix, and examined by scanning electron microscopy. When the HUVECs and Descemets membrane fusion formed a monolayer, the corneal transplantation in rabbits was performed.  Twenty-four New Zealand white rabbits were randomly divided into experimental group and control group (n=12 each), and their left eyes served as recipients. RESULTS：Cultured HUVECs exhibited polygonal shape. More than 90% HUVECs were labeled with CM-DiI and the cell membrane was positive with red fluorescence, which was detectable at least up to 3 generations. The histological examination indicated that porcine cornea cells were clearly extracted, and the collagen fibers were well arranged. A continuous monolayer of HUVECs on the porcine cornea acellular matrix was observed under scanning electron microscopy. The reconstructed corneal posterior lamellae were similar to the normal cornea. The observation of transplantation showed that the cornea in experimental group was substantially transparent. However, that in control group was oedematous and adiaphanous. CONCLUSION：Corneal posterior lamellae can be reconstructed in vitro by cultivating HUVECs on porcine cornea acellular matrix. After xenogeneic transplantation, the graft survives in vivo and expresses normal corneal endothelial cell biological functions. Deep lamellar corneal endothelial transplantation is an effective keratoplasty.