摘要
目的通过体内包埋实验将优化的脱细胞真皮基质(ADM)与成纤维细胞(FB)体外培养,探讨是否具有生物相容性。方法用高渗盐-NaOH消蚀法制备得到大鼠ADM,用低浓度戊二醛交联ADM,未交联A组(0min)作为对照,根据交联时间不同分成B组(10min)、C组(15min)和D组(30min)。通过大鼠体内包埋实验对比各组ADM物理性状、包埋后面积、炎性反应、浸润生长的FB和毛细血管数目,选择出最佳交联时间。制备优化的人ADM,用四甲基偶氮唑盐(MTT)比色法检测ADM的细胞毒性;人FB与ADM复合培养检测其细胞相容性,用免疫组织化学和透射电镜观察FB的生长和增殖情况;反转录聚合酶链反应(RT-PCR)检测FBⅠ、Ⅲ型前胶原mRNA的表达。结果大鼠体内包埋实验显示,4周取材,未交联组不易触及移植物,植入物和周围的软组织基本融合难以分开;未交联组ADM的面积较包埋前缩小;未交联组ADM内有少量炎性细胞,胶原纤维嗜酸性弱;交联组ADM内未见炎性细胞,浸润生长的FB数目、血管数目与戊二醛交联时间的单因素相关性分析呈负相关;最佳的交联时间为10min。体外培养显示,优化ADM的细胞毒性反应0～1级;人FB能够很好地在优化的ADM表面贴附、生长和增殖;ADM内FBⅠ型、Ⅲ型前胶原mRNA的表达降低。结论高渗盐-NaOH消蚀法制备ADM,工艺简单、成本低、生物相容性好,可以提供细胞生长的空间,并可在细胞间转导通讯和信号,下调细胞外基质代谢系统。
Objective To select the best glutaraldehyde cross-linkage by implantation experiment and obtaining the optimizing acellular dermal matrix （ADM） by the method of NaOH maceration and the best glutaraldehyde crosslinkage. Fibroblasts（FBs） were cultured on optimized ADM and observed to find a good biocompatibility. Methods We prepared rat ADM by the method of NaOH maceration. The resulting ADM was divided into four groups by different crosslinkage, including group A （0min） , group B （10min） , group C （15min） and group D （30min）. We compared physical character, area after implantation, inflammation reaction and the number of infiltrated FBs and blood vessels of four groups by ordinary and histological observation in order to select the best cross-linkage. The cytotoxicity of ADM was evaluated by methyl thiazolyl tetrazolium （ MTT ） colorimetry. FBs were cultured on optimized ADM and observed by immunohistochemistry and transimission electron microscope（TME）. The expression of type Ⅰ and Ⅲ precollagen mRNA in the FBs was examined by RT-PCR. Results Four weeks after implantation, areas of uncross-linked ADM were decreased and the ADM closely adhered to the tissue around, inflammation cells could be observed. There were a few inflammation cells and statistical negative correlation between the number of infiltrated cells/blood vessels and cross-linkage in the cross-linked ADM. The best cross-linkage time was 10 min. The eytotoxicity scores of optimized ADM were grade 0 or 1. FBs grew and proliferated well in the optimized ADM. Compared with the one grown on the plate, the expression of type Ⅰ and Ⅱ precollagen mRNA of FBs grown in the optimized ADM were decreased. Conclusion The process of NaOH maceration is simple and low cost. The optimized ADM for 10minutes has good biocompatibility and may provide space and transduct information for cells growth. It effers a molecular biology foundation which may reduce scar formation because of lowly regulating metabolism system of extra cellular matrix （ ECM）.
出处
《解剖学报》
CAS
CSCD
北大核心
2010年第5期768-773,共6页
Acta Anatomica Sinica
