利用海藻酸水凝胶构建仿肝板肝组织三维共培养模型

发布时间：2018-11-23 18:51

【摘要】：目的利用海藻酸水凝胶构建一种新的肝细胞三维共培养模型。方法利用海藻酸钠、微流控芯片,以及肝细胞C3A和脐静脉内皮细胞EA.hy926制备出海藻酸钠水凝胶微纤维,实验组为仿肝板组,同时制备出混合无序水凝胶微纤维作为对照组。利用活细胞双荧光标记验证微纤维内两种细胞排列结构,将微纤维培养1周,每天观察微纤维形态,检测肝细胞活力及清蛋白(Alb)、丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)、乳酸脱氢酶(LDH-L)、α1抗胰蛋白酶(α1AT)、凝血因子Ⅶ(FⅦ)、谷胱甘肽S转移酶α1(GSTα1)、细胞色素P450氧化酶1A2(CYP1A2)的水平。结果仿肝板组水凝胶内C3A细胞在中间,有2~3排,EA.hy926细胞位于C3A细胞两侧,呈现肝板结构排布;对照组水凝胶内两种细胞则混杂在一起呈无序状态;大约3d肝组织条索形成;两组水凝胶微纤维直径随时间变化差异无统计学意义(P0.05);仿肝板组肝细胞活力在第5天达到最大值,对照组在第6天达到最大值,两组除第1天较接近外,其余各天仿肝板组均高于对照组;两组清蛋白分泌水平变化趋势基本相同,在第3天达到最大值,第4天开始下降;仿肝板组ALT、AST、LDH-L在第3天下降到最小值,第4天以后变化趋势和对照组相同;两组α1AT除第5天外其余各时间点比较差异均有统计学意义(P0.05);两组GSTα1分泌量随时间持续上升,仿肝板组各时间点明显高于对照组(P0.05);仿肝板组FⅦ分泌量前7d逐渐升高,对照组于第2天持续下降,仿肝板组第3天开始明显高于对照组(P0.05);对照组细胞内CYP1A2水平随时间变化不明显,仿肝板组从第4天开始明显高于对照组(P0.05)。结论成功构建出一种仿肝板肝组织三维共培养模型,肝细胞功能有望得到长时间维持。
[Abstract]:Objective to construct a new three-dimensional co-culture model of hepatocytes by alginate hydrogel. Methods Sodium alginate hydrogel microfibers were prepared by sodium alginate, microfluidic chip, hepatocyte C3A and umbilical vein endothelial cell (EA.hy926). The double fluorescent labeling of living cells was used to verify the arrangement of two kinds of cells in the microfiber. The microfibers were cultured for 1 week. The morphology of the microfibers was observed, and the viability of the liver cells and the activity of the albumin (Alb), alanine aminotransferase (ALT),) were detected. The levels of aspartate aminotransferase (AST), lactate dehydrogenase (LDH-L), 伪 1-antitrypsin (伪 1AT), coagulation factor 鈪，

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