筛选适宜于构建工程化肝组织的凝胶材料支架

Study on choice of gel-type material stents for constructing engineered liver tissue

目的使用不同可注射性凝胶支架体外进行新生大鼠肝细胞立体培养,为后期体内植入筛选相容性较好的支架材料。方法使用胰酶冷消化法分离新生大鼠肝细胞。将肝细胞分别与纤维蛋白胶、壳聚糖凝胶、鼠尾胶原、透明质酸钠支架材料复合,形成工程化肝组织凝胶,接种于培养板中,并通过定期光镜来观察大鼠肝细胞,四唑盐(MTT)比色法评价细胞活性。溴甲酚绿法测上清白蛋白含量,脲酶比色法测定上清尿素含量。结果四组均可形成三维生长的工程化类肝样组织。培养第4天,生物蛋白胶及壳聚糖组的培养上清液中尿素含量达到高峰,分别为培养第1天的1.31倍和1.28倍,随后含量缓慢下降。培养第7天,新生大鼠肝细胞在纤维蛋白凝胶及壳聚糖凝胶中密集生长,同时MTT显示细胞活性及白蛋白分泌达到高峰,OD值分别为培养初期的1.11倍和1.17倍,上清白蛋白含量分别是初期的1.13倍和1.15倍。而在透明质酸钠及鼠尾胶原凝胶中则细胞生长缓慢,白蛋白及尿素含量持续下降。结论 4种可注射性支架中,壳聚糖和生物蛋白胶对于新生大鼠肝细胞生物相容性较好,透明质酸和鼠尾胶原较差,前两者更适合用于工程化肝组织的构建。

Objective Neonatal rat liver cells were three-dimensional cultured in vitro with different injectable gel stents,to screening better compatibility material for implantation.Methods Neonatal rat liver cells were separated and digested by trypsin digestion method.Cells mixing with fibrin glue,chitosan gel,rat tail collagen,hyaluronic acid scaffolds were seeded in culture plates respectively.The function of newborn rat liver cells was determined in vitro through regular light microscope observation.Cell activity was evaluated by MTT determination.Supernatant albumin and supernatant urea contents were measured by bromocresol green method and urease colorimetry respectively.Results Liver cells showed three-dimensional formation in the four groups.On the fourth day,supernatant urea contents in the fibrin glue and chitosan gel groups reached a peak（1.31 times and 1.28 times respectively）,then reduced gradually.On the seventh day,a dense growth of neonatal rat liver cells showed in fibrin glue and chitosan gel,meanwhile,MTT showed that cell activity and albumin secretion reached a peak,the OD values were 1.11 times and 1.17 times that of the values in early stage.The supernatant albumin contents were 1.13 times and 1.15 times respectively.The growth of cells was slowly in the hyaluronic acid and rat tail collagen groups.Albumin and urea contents also showed consecutive decline.Conclusion Of the 4 kinds of injectable stents,chitosan,and fibrin glue showed better biocompatibility to neonatal rat liver cells,and rat tail collagen and hyaluronic acid are poor.The former two stents are suitable for engineered liver tissue building.