尿道细胞外基质的研制

The preparation of extracellular matrix for the replacement of urethra

目的 探讨尿道细胞外基质 (ECM )的制备方法。 方法 采用 4因素 3水平 ,9次实验的正交设计 [L9(34) ]。切取 37只兔的尿道 ,2 7条按正交设计随机分为 9组行尿道脱细胞处理 ,试验重复 3次。采用HE染色及计算机图像分析对尿道基质进行残余细胞成分计数 ,统计分析获得最佳方法 :0 .4 %胰蛋白酶、1%甲醛加 0 .2 %戊二醛、4 0U/mlDNA酶 (A3 B2 C3 方案 )。另 10条兔尿道制备成尿道ECM并用于同种异体尿道缺损修复实验 ;分别于修复术后 10d ,3、6及 2 4周取材观察缺损修复处组织再生情况。 结果 各组脱细胞后残余细胞成分量均不相同 ,第 7、9组未发现细胞残余成分 ;制备的尿道ECM经扫描电镜分析未发现细胞残片。缺损修复实验术后 10d ,基质中见单层上皮细胞 ,且有血管长入ECM中 ,但管径较小 ,基质和受体尿道连接处有炎性细胞浸润 ;3周时尿道ECM管腔已完全被上皮细胞覆盖 ;6周时可见平滑肌细胞再生 ,炎性细胞消失。 2 4周后基本结构近似正常。 结论 制备尿道细胞外基质中 3个关键条件的最佳水平为A3 B2 C3 ,即在尿道脱细胞处理过程中采用 0 .4 %胰蛋白酶、1%甲醛加 0 .2 %戊二醛、4 0U/mlDNA酶。

Objective To evaluate an ideal way to prepare the extracellular matrix of urethra. Methods An orthogonal design [L9(34)] was used in the experiment.Urethras were obtained from 37 rabbits,among which 27 segments were randomly selected and were decellularized following the orthogonal design in 9 groups.The whole experiments were repeated for 3 times.After the decellularization process,the acellularity of the ECM was examined by haematoxylin-eosin staining.The optimum way was found out through comparing the numbers of the remained cellular elements by computer image analysis.An ideal way was found by statistic analysis.Then the ECM was obtained from 10 pieces of urethras by the optimum methods.The scanning electronic microscopy was used to confirm the decellulary matrix.Subsequently,the ECM was used as a graft for replacement. In 10 rabbits,the urethral defect were replaced with the urethral ECMs. At sacrifice,10 days,3 weeks,6 weeks and 24 weeks,the grafts was taken out,and the regeneration was confirmed by the haematoxylin-eosin staining. Results ECM resulting from different dedellularization process in the urethras are different in the numbers of remaining cellular elements.There are no cellular elements in the 7th and the 9th group of the tissues.The cellular elements was not found by the scanning electronic microscopy in the ECM getting from the optimum methods.In the animals with replacement,histologic examination showed complete regeneration 24 weeks post operation. Conclusions The best way to prepare the ECM of urethra is A 3B 2C 3.