一种新型脱细胞猪角膜基质载体支架的制备及其鉴定研究

发布时间：2018-06-20 23:53

本文选题：脱细胞猪角膜基质 + 载体支架　；参考：《山东大学学报(理学版)》2017年05期

【摘要】：为了获得基于异种角膜材料的理想组织工程角膜载体支架,首次建立了脱氧胆酸钠(SD)和原钒酸钠(SO)联合处理的技术方法,利用新鲜猪角膜进行了脱细胞角膜基质(aPCS)的制备及其鉴定研究。用角膜板层刀从新鲜猪角膜中切下厚度450μm的角膜片,分别选用SD联合SO、十二烷基磺酸钠(SDS)和Triton X-100的去细胞处理方法制备出SD-aPCS、SDS-aPCS和Triton-aPCS共3种支架,利用外观照相、分光光度计、石蜡切片苏木紫-伊红(HE)染色、冰冻切片DAPI和阿利新蓝染色检测了其理化性质和组织结构;对SD-aPCS进行扫描和透射电镜鉴定后,进而利用噻唑蓝(MTT)、石蜡切片HE染色、冰冻切片DiI荧光观察以及免疫荧光细胞化学染色评估了其对非转染人角膜基质(ntH CS)细胞的毒性与生物相容性。检测结果发现,3种aPCS的细胞脱除干净且在干重和含水量上没有显著差异;其中,SD-aPCS的透明性与糖胺聚糖(GAG)含量最高、SDS-aPCS次之、Triton-aPCS最低;除Triton-aPCS的组织结构出现了明显的紊乱外,SD-aPCS和SDS-aPCS的组织结构均排列规则。在电镜下,SD-aPCS的前弹力层表面平整、无裂痕,板层结构和胶原纤维超微结构正常;此外,SD-aPCS浸提液对ntH CS细胞没有毒性作用,注射接种到SD-aPCS支架内的ntH CS细胞与支架嵌合紧密,随体外培养时间的延长而逐渐伸展和迁移,且细胞仍保持有其固有标志蛋白—波形蛋白,细胞连接蛋白—间隙连接蛋白-43和整联蛋白,以及膜运输蛋白—钠钾泵的阳性表达。由此可见,利用SD联合SO的方法所制备SD-aPCS具有理想的理化性质、组织结构和生物相容性,可作为一种理想的载体支架用于组织工程角膜的体外构建及其相关应用研究。
[Abstract]:In order to obtain an ideal scaffold for corneal tissue engineering based on xenogeneic corneal materials, a new method of combined treatment of sodium deoxycholate and sodium orthovanadate (SOO) was established for the first time. The preparation and identification of acellular corneal stroma (APCS) from fresh porcine cornea were studied. Using lamellar knife to cut 450 渭 m corneal slices from fresh porcine cornea, SD combined with SO, SDS and Triton X-100 were used to prepare SD-aPCS SDS-aPCS and Triton-aPCS scaffolds, respectively. SD-aPCS and Triton-aPCS were used to produce SD-aPCS and Triton-aPCS respectively. The physicochemical properties and histological structure of paraffin sections were examined by DAPI and Alisin blue staining, and then the SD-aPCS were identified by scanning and transmission electron microscopy, and then the paraffin sections were stained with thiazolyl blue (MTT) and paraffin sections by HE staining. The toxicity and biocompatibility of frozen sections of human corneal stromal cells were evaluated by fluorescence observation and immunofluorescence cytochemical staining. The results showed that the cells of the three kinds of APCS were removed clean and had no significant difference in dry weight and water content, the transparency of SD-aPCS and the content of glycosaminoglycan (GAG) were the highest, and the content of SDS-aPCS was the lowest, and that of Triton-aPCS was the lowest. The organization structure of SD-aPCS and SDS-aPCS were arranged regularly except for the obvious disorder in the organizational structure of Triton-aPCS. Under electron microscope, the surface of the anterior elastic layer of SD-aPCS was flat, without cracks, the lamellar structure and the ultrastructure of collagen fibers were normal, in addition, the extract of SD-aPCS had no toxic effect on NTH CS cells, and the NTH CS cells injected into SD-aPCS scaffold were closely associated with the scaffold chimerism. With the extension of culture time in vitro, the cells gradually extended and migrated, and the cells still maintained the positive expression of vimentin, connexin 43 and integrin, as well as membrane transport protein-sodium potassium pump. It can be seen that SD-aPCS prepared by the method of SD combined with so has ideal physicochemical properties, tissue structure and biocompatibility, and can be used as an ideal carrier scaffold for in vitro construction of tissue engineered cornea and its related applications.
【作者单位】：中国海洋大学海洋生命学院角膜组织工程重点实验室;
【基金】：国家高技术研究发展计划(863计划)资助项目(2006AA02A132)
【分类号】：R318.08;R779.65

【相似文献】