脱细胞猪膀胱支架的构建

Construction of acellular porcine bladder scaffolds

背景:膀胱修补术是目前临床上治疗膀胱缺损的主要方法之一,同源性组织因各种因素的影响来源较少,组织工程膀胱脱细胞基质受到人们越来越多的关注。猪膀胱脱细胞外基质来源广泛,具备天然的细胞外支架结构,成为组织工程膀胱替代材料研究的热点。目的:研究脱细胞猪膀胱作为组织工程支架材料的可行性。方法:取新鲜的猪膀胱,联合液氮冻融、十二烷基硫酸钠、胰蛋白酶脱细胞方法制猪膀胱无细胞基质。按不同脱细胞方法分组:(1)正常对照组:不做任何处理;(2)实验组:0.6%胰蛋白酶+5%十二烷基硫酸钠(pH 8.0);(3)脱细胞对照组:分别用0.75%胰蛋白酶(pH 8.0)、1%胰蛋白酶(pH 8.0)、5%十二烷基硫酸钠(pH 7.6)或10%十二烷基硫酸钠(pH 7.6)处理。通过苏木精-伊红染色和VG染色、DNA定量、α-Gal抗原检测,观察猪膀胱脱细胞效果。结果与结论:苏木精-伊红染色显示实验组猪膀胱细胞成分已基本去除;VG染色显示实验组完整保留了猪膀胱组织的细胞外基质成分;实验组的DNA残留量仅为(49.84±30.13)μg/g,显著低于几个脱细胞对照组(P<0.05);同时其α-Gal抗原残留也明显低于脱细胞对照组。提示:应用0.6%胰蛋白酶+5%十二烷基硫酸钠处理猪膀胱,在完整保留猪膀胱组织细胞外基质的同时,可有效去除其细胞成分,为构建脱细胞猪膀胱支架提供一定参考价值。

BACKGROUND: Bladder repair is currently one of the main treatments for bladder def ects. Homologous tissue is less affected by various factors. Tissue engineered acellular bladder matrix has become an increasing area ofinterest. Porcine bladder acellular matrix has a wide range of sources and has a natural extracellular scaffold struc ture, which has become a hot topic in tissue engineering bladder substitute materials. OBJECTIVE: To explore the feasibility of acellular porcine bladder as a tissue engineering scaffold material. METHODS: The cell-free matrix of pig bladder was prepared by liquid nitrogen freezing and thawing, dodecyl sodium sulfate and trypsin decellularization method. According to different decellularization methods, pig bladders were divided into normal control gro up(without any treatment), experimental group(treated with 0.6% trypsin and 5% sodium lauryl sulfate(pH 8.0)) and acellular control group(treated with 0.75% trypsin(pH 8.0), 1% trypsin(pH 8.0), 5% sodium lauryl sulfate(pH 7.6) or 10% sodium dodecyl sulfate(pH 7.6)). The d ecellularization effect of pig bladder was observed by hematoxylin-eosin staining, van Gieson staining, DNA quantification, and α-Gal antigen detection. RESULTS AND CONCLUSION: Hematoxylin-eosin staining revealed that in the experimental group, the components of the bladder cells of the pigs were basically removed. van Gieson staining revealed that the DNA residues and α-Gal antigen residues in the cells were significantly lower than those in the control group(P<0.05). These results suggest that treatment of pig bladder with 0.6% trypsin and 5% sodium dodecyl sulfate can effectively remove its cellular components while retaining the extracellular matrix of porcine bladder tissue. This provides a reference value for constructing accelular porcine bladder scaffolds.