脱细胞化肝脏生物衍生支架的制备及鉴定  

Preparation and identification of hepatic decellularized bio-derived scaffold

作　　者：康玉占[1] 汪艳[1] 高毅[1] 

机构地区：[1]南方医科大学附属珠江医院肝胆二科,南方医科大学组织工程与再生医学研究所,广东省广州市510282

出　　处：《中国组织工程研究与临床康复》2009年第8期1505-1508,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家高技术研究发展计划(863计划)(2006AA02A141);广东省自然科学基金(7300180)~~

摘　　要：背景:肝细胞在体外迅速失去极性及合成代谢障碍成为肝脏组织工程研究需要克服的一个难题,在体外寻找一个有利于肝细胞生长与功能维持的细胞外基质微环境成为目前研究的焦点。目的:制备肝脏去细胞化细胞外基质生物衍生支架,并对该生物衍生支架进行初步鉴定。设计、时间及地点:观察性实验,于2008-02-15/05-01在广东省脑功能修复与再生研究所完成。材料:10只雄性SD大鼠用于制备去细胞肝脏细胞外基质支架。10只雄性SD大鼠用于制备原代肝细胞。方法:将SD大鼠肝脏切成10mm×5mm的组织片后,经过胰酶-乙二胺四乙酸作用24h、去垢剂曲拉通×10072h后得到肝脏去细胞化生物衍生支架。每只大鼠获取2×108个肝细胞,将所得原代细胞与肝脏去细胞化生物衍生支架放入含DMEM-F12,体积分数为10%胎牛血清,胰岛素0.5U/mL,地塞米松1×10-7mmol/L,表皮生长因子10μg/L的培养基中共培养,并与单纯原代细胞培养相比较。主要观察指标:培养14d时组织块行苏木精-伊红染色,Masson染色进行组织学分析。并行扫描电镜观察。对培养1,3,5,7,9,14d上清液进行白蛋白及尿素水平检测。结果:组织学检查未见明显细胞核残存,大量胶原纤维得到保留,扫描电镜示纤维成网状排列。支架与原代肝细胞共培养上清液白蛋白和尿素水平高于单纯原代肝细胞培养(P<0.05)。结论:利用去垢剂与胰酶-乙二胺四乙酸低渗溶液处理可以有效完整去除肝脏组织块的细胞成分,较完整地保留细胞外基质;该生物衍生支架有利于肝细胞的生长及功能维持。BACKGROUND： Losing polarity rapidly in vitro and disorder of synthesis and metabolism of hepatocytes are difficult problems which need to be overcome in hepatic tissue engineering. It has become a focus to look for an extracellular matrix microenvironment which is benefit for maintaining growth and function of hepatocytes. OBJECTIVE： To prepare hepatic decellularized extracellular matrix bio-derived scaffold, and to perform preliminary identification. DESIGN, TIME AND SE＇B＇ING： An observational experiment was performed in Guangdong Provincial Institute of Cerebral Function Repair and Regenerative Medicine from 15th February to 1st May in 2008. MATERIALS： Ten male Sprague Dawley （SD） rats were used to prepare hepatic decellularized extracellular matrix bio-derived scaffold. The other ten male SD rats were used to prepare primary generation hepatocytes. METHODS： The liver from 10 rats were cut into 10 mmx5 mm tissue slices followed by treatment with trypsin-0.02%EDTA for 24 hours and then 1%TritonX100 for 72 hours to prepare hepatic decellularized bio-derived scaffolds were prepared. Hepatocytes of primary generation were obtained from the other ten rats （2×10^8 hepatocytes from each rat）. The primary hepatocytes were cocultured with prepared hepatic decellularized bio-derived scaffolds in DMEM-F12 medium containing 10% fetal bovine serum ＋0.5 U/mL insulin ＋ 1×10^-7 mmol/L dexamethasone ＋ 10 μg/L epidermal growth factor. Comparison between scaffold coculture and simple primary culture was performed. MAIN OUTCOME MEASURES： Hematoxylin-eosin staining and Masson staining were performed at 14 days after culture for histological analysis under a scanning electron microscope. The levels of albumin and urea in supernatant were detected at 1, 3, 5 7, 9, and 14 days after culture. RESULTS： Histological evaluation revealed that no obvious nucleus was remained but a lot of collagen fibers were preserved. The preserved fibers showed a network arrangement under the scanning electron microscope

关 键 词：脱细胞化 肝组织块 细胞外基质 肝组织工程 

分 类 号：R318[医药卫生—生物医学工程]