去细胞全肾生物支架的制备与鉴定  

Preparation and identification of a whole kidney decellularized bio-derived scaffold

作　　者：张建色[1] 王辉[2] 邵培刚[2] 林贤丰[2] 彭蒙蒙[2] 薛向阳[3] 梅劲[1] 

机构地区：[1]温州医学院人体解剖学教研室 [2]温州医学院第一临床医学院 [3]温州医学院微生物与免疫学教研室,浙江温州325035

出　　处：《解剖学报》2012年第2期253-257,共5页Acta Anatomica Sinica

基　　金：国家自然科学基金资助项目(81071576);浙江省教育厅资助项目(Y20106491);温州市科技局资助项目(Y20100012);浙江省新苗计划资助项目(2011R413015)

摘　　要：目的通过灌注加浸泡法制备全肾细胞外基质支架,并对该生物支架进行鉴定。方法健康成年SD大鼠20只,取肾,行肾动脉留置针插管,灌注压3.6mmHg,恒温37℃依次浸泡灌注肝素化PBS溶液、0.05%胰蛋白酶溶液、1%十二烷基硫酸钠(SDS)溶液、1%Trition X-100溶液以及含青、链霉素的PBS溶液。处理后,行DNA定量与定性分析,透射电镜、HE染色及免疫荧光观察残留细胞核成分,进一步丙烯腈-丁二烯-苯乙烯树脂(ABS)铸型观察肾内血管分布情况。结果去细胞组DNA含量较对照组下降了97%,琼脂糖凝胶电泳未见明显DNA条带;透射电镜、HE染色及免疫荧光观察去细胞组保留了大量细胞外基质,未见明显细胞核成分残留;铸型标本显示去细胞组血管较稀疏,分支完整、清晰。结论联合酶消化法与去垢剂洗涤法,经灌注加浸泡法处理的全肾,可有效清除肾内所有细胞成分,较好地保留细胞外基质支架和血管网络结构,是一种简单易行且较为理想的制备组织工程肾生物支架的方法。Objective To prepare a whole kidney decellularized extraeellular matrix bio-derived scaffold and to perform preliminary identification. Methods The kidney with ureters and renal vessels was harvested from adult SD rats. Intravenous catheters were inserted through the renal arteries to establish channels for whole-kidney perfusion successively with heparinized PBS,0.05% trypsin, 1% Sodium dodecyl sulfate（SDS）, 1% Triton X-100 and antibiotic-containing PBS under a pressure of 3.6 mmHg in 37℃. After deeellularization,the scaffold and native kidney were observed through genomic DNA content analysis, transmission electron microscopy, HE staining, immunofluorescence and vascular cast. Results Quantitative analysis of DNA content within the scaffold showed a 97% decrease compared to the native kidney. The agarose gel electrophoresis showed no DNA bands associated with the decellularized scaffold. Transmission electron microscope, HE and immunohistoehemistry showed a lot of collagen fibers but no visible cell nuclei remained after decellularization. Cast specimen showed that renal arteries were more sparse ,but still full and clear compared with the native kidney. Conclusion The method of combined enzymatic digestion and detergent washing,soaking through the infusion is a simple,ideal preparation for the bioengineering scaffold of the kidney,which enables the quick and consistent removal of all the cellular components of the tissue,leaving behind mostly intact the extracellular matrix elements and an integrated vascular network.

关 键 词：去细胞 肾 细胞外基质 血管铸型 组织工程 免疫荧光 大鼠 

分 类 号：R322[医药卫生—人体解剖和组织胚胎学]