灌注法制备大鼠全肾脏脱细胞基质支架的细胞相容性

Cellular biocompatibility of whole-kidney acellular matrix in rats by perfusion

背景:作者所查目前尚未见应用灌注法制备大鼠全肾脱细胞基质的相关报道,制备的脱细胞基质是否具有良好的细胞相容性尚不确切。目的:应用灌注法制备大鼠全肾脏脱细胞基质支架,检测鼠肾脱细胞基质与L929细胞的细胞相容性,探讨灌注法制备的肾脱细胞基质作为细胞支架构建泌尿系实质性组织工程器官的可行性。设计、时间及地点:体外细胞水平观察实验,于2009-02/05在南方医科大学珠江医院中心实验室完成。材料:取12周龄Wistar大鼠的肾脏,保留输尿管、肾静脉及肾动脉,沿肾动脉插入留置针建立灌注通道。灌注压强为9.81kPa,依次灌注肝素化PBS溶液,1%十二烷基磺酸钠溶液,去离子水,1%TritonX-100溶液以及含青链霉素的PBS溶液,制备全肾脱细胞基质支架。方法:①设立空白组(无细胞)、阴性对照组(培养基)和实验组(鼠肾脱细胞基质浸提液)及阳性对照组(含0.64%苯酚溶液的培养基)。取对数生长期的L929细胞,4×103/孔接种于96孔板中,每组各5孔,于接种24,72,120h,通过MTT法显色,于酶标仪490nm波长下检测吸光度值,计算相对增殖率。②设立对照组(培养基)、实验组(鼠肾脱细胞基质浸提液)及阳性对照组(含0.64%苯酚溶液的培养基),培养48h后应用流式细胞技术检测细胞凋亡情况。主要观察指标:①支架微观结构。②细胞毒性。③细胞凋亡率。结果:脱细胞基质支架扫描电镜观察仅见基膜及胶原蛋白等细胞外基质形成网状结构而无细胞残留,24,72,120h实验组吸光度值与阴性对照组比较差异无显著性意义(P>0.05)。脱细胞基质细胞毒性为0级和1级。实验组与阴性对照组之间细胞凋亡率差异无显著性意义(P>0.05)。结论:大鼠全肾脏脱细胞基质支架具有良好的细胞相容性。

BACKGROUND： At present, there is little related report about producing a whole-kidney acellular matrix （ACM） scaffold in rats using perfusion. The cellular biocompatibility of the ACM is poorly understood. OBJECTIVE： To produce a whole-kidney ACM scaffold in rats by perfusion, to evaluate the cytocompatibility of ACM with the L929 cells in vitro, and to assess the possibility of ACM as the cytoskeleton and tissue-engineered urinary organ construction. DESIGN, TIME AND SETTING： An in vitro observation was performed at the Central Laboratory of Zhujiang Hospital, Southern Medical University from February to May 2009. MATERIALS： Kidneys were obtained from 12-week-old Whista rats, while ureter, renal veins and renal artery were reserved. Intravenous catheters were inserted through renal arteries to establish channels for perfusion. Whole-kidney retrograde perfusion was performed with successively heparinized PBS, 1% SDS, deionized water, 1% TritonX-100 and antibiotic-containing PBS under a pressure of 9.81 kPa to prepare whole-kindney acellular matrix scaffolds. METHODS： ① Samples were randomly divided into blank group （without any cells）, negative control group （culture media）, experimental group （rat kidney ACM leaching liquor）, and positive control group （culture media containing 0.64% phenol）. L929 cells in the logarithmic phase were seeded in 96-well plates at the density of 4× 10^3/well, with 5 wells in each group. At 24, 72, and 120 hours after incubation, cells were stained with MTT method to detect absorbance at 490 nm and calculate relative growth rate. ② Control group （culture medium）, experimental group （rat kidney ACM leaching liquor）, and positive control group （culture media containing 0.64% phenol） were set up to detect cell apeptosis at 48 hours after culture using flow cytometry. MAIN OUTCOME MEASURES： Microstructure of the scaffold, cytotoxicity and cell apoptotic rate. RESULTS： After SDS and TritonX-100 union processing, reticulate structures made of basilar membrane and collagen were shown under scanning electron microscope rather than normal structures of cells. At every time intervals （24, 72, and 120 hours）, there was no significant difference in the absorbance between experimental group and negative control group （P 〉 0.05）. The grade of the cytotoxicity of the ACM was .0-1. There was no significant difference in cell apoptotic rate between experimental group and negative control group （P 〉 0.05）. CONCLUSION： The whole-kidney acellular matrix scaffolds in rat by perfusion have good biocompatibility.