去细胞猪主动脉瓣的构建及性能鉴定  

Construction and evaluation of the property of decellular porcine aortic valve

作　　者：刘维永[1] 金振晓[1] 顾春虎[1] 赵东锷[1] 谭红梅[1] 

机构地区：[1]第四军医大学附属西京医院心胸外科,西安710032

出　　处：《中华医学杂志》2005年第5期324-327,共4页National Medical Journal of China

基　　金：国家自然科学基金资助项目(30271292)

摘　　要：目的研究猪主动脉瓣(猪瓣)去细胞方法,观察埋藏前后猪瓣内源性逆转录病毒(PERV)和瓣膜支架变化.方法①采用去垢剂、核酸酶和改变溶液渗透压方法制备去细胞猪瓣支架.②应用聚合酶链式反应(PCR)和逆转录-聚合酶链式反应(RT-PCR)等方法检测去细胞前后新鲜猪瓣和去细胞猪瓣以及供体和受体外周血PERV.③去细胞瓣叶埋藏家兔皮下4、6、8、10周后分别取材,应用苏木素-伊红和免疫组织化学染色,Instron 1122型万能强力仪、分光光度计、光镜和电镜进行检查.结果①经去细胞处理后猪瓣内细胞成分完全去除,可溶性蛋白明显丢失[(0.24±0.04)% vs (0.48±0.12)%],瓣叶含水量增加[(92.16±1.48)% vs (89.20±1.55)%],P均＜0.01,但去细胞猪瓣仍保持了良好纤维支架结构,热皱缩温度[(72.0±0.7)℃ vs (71.2±0.8)℃]和断裂强度[(449±19)g/mm2 vs (541±19)g/mm2]均无明显变化.②新鲜猪瓣和猪外周血标本经PCR和RT-PCR检测,于琼脂糖凝胶电泳内均出现219 bp扩增带,经测序这一产物与Medline公布的PERV-C型病毒有90%～97%同源性;去细胞猪瓣,及其移植于犬体内一个月后,抽取犬的外周血标本检测,219 bp扩增带均消失.③埋藏于兔皮下的去细胞猪主动脉瓣有轻微组织反应,4周时可见中性粒细胞、淋巴细胞和浆细胞浸润;6周后上述炎性细胞明显减少,去细胞瓣叶周边部分开始吸收,10周后完全吸收,埋藏部位未见结缔组织增生或疤痕形成.结论①猪瓣内细胞成分可以完全去除,并保持良好的纤维支架结构和机械强度,抗原性轻微,是一种生物可降解性材料.②猪瓣存在C型PERV,当瓣叶细胞完全去除后PERV-C转为阴性,受体外周血标本PERV-C反应为阴性.Objective To construct decellular porcine aortic valve (PAV) and to observe the existence of porcine endogenous retrovirus (PERV) and valve scaffold structure before and after implantation. Methods ① Porcine aortic valve was obtained. The cellular components of PAV were completely removed by using detergent and nucleotidase solution combined with alteration of osmosis. ② The decellular underwent HE staining and light microscopy and detection of its physical and chemical properties. ③ 20 pieces of decellular PAV were implanted into dogs. On e month later blood samples of the dogs were collected. PCR and RT PCR were used to detect the PERV expression in 20 samples of pig′s peripheral blood, 20 fresh PAVs, cultured pig kidney cells of the PK15 line (as positive control), decellular PAVs implanted into the dogs, and 10 samples of dogs' peripheral blood. ④ Small pieces of decellular PAVs were implanted into the subcutaneous tissues of 6 rabbits at the back, 6 pieces for one rabbit, and then extracted by the ends of the 4 th , 6 th , 8 th and 10 th week respectively after implantation to undergo HE staining and light microscopy. Results ① Almost all cellular components in the PAVs had been removed after decellularization; the soluble protein contents lost markedly [(0.238±0.038)% vs. (0.484± 0.116 )%]; the water content of the decellular tissues increased significantly [(92.16±1.48)% vs. ( 89.2 ±1.55)%]; however, the decellular PAVs still maintained their excellent fibrous scaffold structure, and their shrinkage temperature and tension at fracture were not significantly changed [(72.0±0.7)℃ vs. (71.2±0.8)℃, and (448.7±18.65)g/mm 2 vs. (540.7±19.46)g/mm 2 respectively]. ② Agarose gel electrophoresis of all fresh PAVs and porcine peripheral blood samples showed a 219 bp band, which was 90% to 97% homologous with PERV C gene, and the sequence of which is published in Medline. No 219 bp amplified band was found in all decellular PAVs and the peripheral blood sample

关 键 词：去细胞 主动脉瓣 外周血 血标本 皮下 瓣叶 纤维支架 埋藏 吸收 可溶性蛋白 

分 类 号：R318.1[医药卫生—生物医学工程]