超高压脱细胞技术制备组织工程带瓣血管支架  

Tissue-engineered blood vessel scaffolds with valves prepared by hyperpressure acellular technique

作　　者：殷猛[1] 刘锦纷[1] 藤里俊哉 凑谷谦司 中谷武嗣 

机构地区：[1]上海交通大学医学院上海儿童医学中心胸外科,上海市200127 [2]日本国立循环器病中心研究所再生医学部、脏器移植部,日本大阪府吹田市

出　　处：《中国组织工程研究与临床康复》2008年第19期3605-3608,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：中国卫生部笹川医学奖学金;日本国厚生劳动省科学研究费~~

摘　　要：背景：研究表明同种瓣膜移植术后发生的退行性变性与其留存的细胞成分激发宿主免疫反应有显著的相关性。目的：采用超高压结合核酸酶洗涤方法处理猪带瓣膜血管。观察瓣膜脱细胞的效果及猪内源性反转录病毒的去除情况。设计、时间及地点：对比观察实验。于2004-04／2005-04在日本国立心血管病中心研究所完成。材料：实验选用月龄1-3个月的健康雄性迷你猪幼猪，体质量3-5kg。方法：无菌条件下取出猪带瓣肺动脉血管。分为3组：对照组：无任何处理。表面活性剂组：将带瓣管道用Triton X-100溶液浸渍、搅拌24h后洗净。超高压处理组：用超高压设备以981MPa超高静水压（4℃）将供体来源细胞压碎。结合核酸酶的消化作用，磷酸盐缓冲液的搅拌洗涤，脱去细胞残片形成带瓣膜血管支架。主要观察指标：光镜下观察支架形态结构，透射电镜下观察支架细胞成分去除和支架纤维保留情况。扫描电镜观察支架表面的超微结构。观察两种方法处理瓣膜的弹性率差异。检测猪内源性反转录病毒前病毒DNA。定量检测超高压脱细胞前后带瓣管道中DNA含量的变化。结果：①超高压处理组瓣叶及瓣根组织表面及内部细胞完全消失。保留了细胞外基质，DNA含量显著降低。表面活性剂组瓣根组织深部的细胞无法渗透、除去。②超高压处理组瓣膜弹性率几乎不变。瓣膜功能保持较好。表面活性剂组瓣膜弹性率增加。③超高压处理组猪内源性反转录病毒被成功灭活，无法测出。表面活性剂组无法将猪内源性反转录病毒灭活。结论：采用超高压脱细胞方法可基本除去支架内细胞成分，可将内源性反转录病毒成功灭活。保持了细胞外基质结构和功能的完整性，脱细胞效果优于表面活性剂方法。BACKGROUND： Retrograde degeneration after homogenic valvotransplantation is significantly correlated to host immune response activated by remaining cell components. OBJECTIVE： Porcine vessels with valve were managed by ultrahigh pressure combined with nuclease to observe the acellular valve effect and porcine endogenous retrovirus removal DESIGN, TIME AND SETTING： The control observation experiment was performed at the National Cardiovascular Center of Japan from April 2004 to April 2005. MATERIALS： 1-3 mouths old healthy male minipig weighing 3-5 kg were used in this study. METHODS： Pulmonary arterial vessels were sterilely isolated from minipig and divided into three groups, Pulmonary arterial vessels in the control group were intact. Pulmonary arterial vessels in the surface-active agent group were immersed in Triton X-100 solution, stirred and cleaned 24 hours later, Pulmonary arterial vessels in the hyperpressure group treated by cold isostatic pressing （CIP） at 4 ℃ under 981 MPa for disruption of donor-derived cells. The cell debris was then washed out in phosphate buffer saline （PBS） and nuclease, MAIN OUTCOME MEASURES： Vessel stent morphous was observed under a light microscope. Cell component removal and stent fiber reservation were recorded with a transmission electron microscope. Stent ultrastructure was checked with a scanning electron microscope. Differences in elastic rate of valve were measured by two methods. Provirus DNA of endogenous retrovirus was detected. DNA content in vessels was examined before and after hyperpressure. RESULTS： Cells on surface and inner valve leaflet and valve root disappeared in the hyperpressure group, but extracellular matrix was remaining. DNA content was significantly degraded in the treated tissue. Cells in deep valve leaflet could not be removed in the surface-active agent group. Elastic rate and function of the valve nearly did not change in the hyperpressure group. Elastic rate of the valve increased in the surface-active agent group. Porc

关 键 词：同种带瓣血管 支架 脱细胞 猪内源性反转录病毒 超高压 生物材料 

分 类 号：R318[医药卫生—生物医学工程]