膀胱细胞外基质的研制

目的 :探讨膀胱细胞外基质 (ECM)的制备方法。方法 :采用正交设计方法 L9(34) ]。取 1 9只兔的膀胱共 1 9个 ,其中 9个被分成 2 7片 ,按正交设计随机分为 9组行脱细胞处理 ;所有试验重复 3次。经脱细胞处理后 ,采用HE染色及计算机图像分析对基质进行残余细胞成分计数 ,统计分析获得最佳方法。另 1 0个膀胱采用最佳方法制备成膀胱ECM并用于同种异体膀胱缺损修复实验 ;分别于修复术后 1 0d、4周及 3个月取材分析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 ,但第 7组未发现细胞残余成分 ;采用最佳方案(A3 B2 C3 )制备的膀胱ECM经扫描电镜分析未发现细胞残片。缺损修复实验后 1 0d移行上皮组织爬满基质内表面 ;术后 4周可见基质内细胞成分明显增多 ,但较正常组织稀疏 ,排列不规则 ;3个月后 ,取材的组织学分析表明细胞外基质替代部分组织再生状况良好。结论 :制备膀胱细胞外基质的最佳方案是A3 B2 C3 ,即在膀胱脱细胞处理过程中采用 0 .4 %胰蛋白酶、1 %甲醛 +0 .2 %戊二醛、4 0U·ml-1 DNase。

Preparation of Extracellular Matrix for Replacement of Bladder

Objective: To evaluate an ideal way to prepare the extracellular matrix(ECM) of bladder. Methods: An orthogonal design L9(3 4)] concerning three factors which contributes to the preparation of ECM was used in the experiment. Bladders were obtained from 19 rabbits. Nine pieces of bladders were divided into 27 segments and were decellularized according to 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 reached through the comparing of the numbers of the remain cellular elements by computer image analysis. Then the ECM was obtained from 10 pieces of bladders by the optimum method. The scanning electronic microscopy was used to confirm the decellulary. Subsequently, the ECM was used as a graft for replacement. In 10 rabbits, the bladder defect was made and replaced with the bladder ECMs. The grafts were took out 10 days, 4 weeks and 3 months after operation. And the regeneration was confirmed by the haematoxylin eosin staining.Results: ECMs acquired through different decellularization process in the bladders were different in the numbers of remain cellular elements. There were no cellular elements in the 7 th group of the tissues. The cellular elements wasn’t found by the scanning electronic microscopy in the ECM acquired through the optimum method. In all the animals in the replacement, the histologic examination showed complete regeneration 3 months post operation.Conclusion: The best way to prepare the ECM of bladder is A 3B 2C 3.