人工角膜载体的体外构建及生物相容性研究

目的应用壳聚糖(chitosan,CS)和蚕丝蛋白(silkfibroin,SF)构建组织工程角膜基质,探讨不同比例的CS和SF共混膜的生物特性,为以后的角膜移植奠定基础。方法将SF和CS按体积比3GA6FA1和4GA6FA1混合,体外构建具有相应器官特征的角膜基质层;体外培养原代兔角膜基质及上皮细胞,检测细胞分布情况和载体膜片的物理特性;免疫组织化学法测定细胞表型和分布情况,CCK-8试剂盒测定载体膜片毒性,电镜下观察细胞结构形态和附着情况,将构建的膜片植入兔板层角膜中检测组织生物相容性。结果载体膜片的吸水率均在90%左右,明显强于天然角膜,抗拉力SF/CS3GA6FA1组为0.90±0.02,SF/CS4GA6FA1组为1.30±0.05,透光率均大于60%,且抗拉力和透光率随着CS含量增加而增强,但与天然角膜组织间仍存在差异。与载体膜片共培养的细胞在培养2周后,电镜下可见膜片上形成致密的细胞层,重构的板层角膜结构与天然角膜相似。CCK-8法检测结果显示,合成膜无明显毒性。免疫组织化学染色结果显示,培养出的基质细胞形态与天然角膜相似。动物板层移植术后,裂隙灯观察载体膜片组术后半个月内充血消失,无明显的前房炎症反应,术后1个月拆线时无明显新生血管。免疫组织化学法检测术后病理组织未发现CD4+、CD8+、CD31+T细胞浸润。结论CS-SF可以用于组织工程角膜基质的构建,这为重建角膜基质提供了方向。

Construction of artificial cornea stent in vitro and its biocompatibility

Objective　To establish tissue engineered corneal stroma with chitosan (CS) and silk fibroin (SF) and explore the biological characteristics of CS and SF blend membranes with different proportions for corneal transplantation in the future.Methods　SF and CS were combined at 3∶1 and 4∶1 to construct corneal stroma with corresponding organ characteristics in vitro.The primary rabbit corneal stroma and epithelial cells were cultured in vitro for the detection of cell step and physical properties of carrier membrane.Immunohistochemical methods was used to determine cell phenotype and distribution,while CCK-8 kit for cytotoxicity determination.The morphology and adhesion of the cells were observed with electron microscopy.The constructed diaphragm was implanted into the lamellar cornea of rabbits to detect the biocompatibility.Results　The water absorption of the carrier diaphragm was about 90%,which was significantly stronger than that of the natural cornea.The tensile strength of the carrier diaphragm was 0.90±0.02 in the SF/CS 3∶1 group and 1.30±0.05 in the SF/CS 4∶1 group,and the transmittance of the carrier diaphragm was over 60%.The tensile strength and transmittance increased with the increase of CS content,but there were still differences between the carrier diaphragm and the natural corneal tissue.After cultured for 2 weeks with the carrier membrane,cells had a dense cell layer on the membrane with electron microscope,and the reconstructed lamellar corneal structure was similar to that of the native cornea.CCK8 showed no obvious toxicity of synthetic membranes.Immunohistochemical results showed that the morphology of cultured stromal cells was similar to that of natural cornea.After lamellar transplantation,the slit lamp showed the blood congestion disappeared within half a month after operation,and there was no obvious inflammation reaction in the anterior chamber.There was no obvious neovascularization at the time of thread removal one month after operation.No infiltration of CD4+,CD8+,CD31+T cells was detected by immunohistochemistry.Conclusion　It is found that CS-SF can be used to construct tissue-engineered corneal stroma,which can provide direction for corneal stroma reconstruction.