仿肝板肝组织三维共培养对肝细胞功能的影响

发布时间：2018-03-06 17:05

  本文选题：生物人工肝　切入点：海藻酸　出处：《南方医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景肝衰竭是指受到多种因素(如病毒、酒精、药物等)造成肝细胞大量坏死,导致肝脏功能发生严重障碍或失代偿,进而出现以凝血机制障碍、黄疸、肝性脑病、腹水等为主要表现的一组临床症候群。目前肝移植是治疗肝衰竭的有效方法,然而世界各国的器官移植面临着严重的来源短缺问题。为此,以实现肝脏功能修复、替代、重建为目标的人工肝技术和肝组织工程为肝衰竭治疗提供了新的选择。研究目的和意义:我们通过结合肝组织工程与生物人工肝技术,模拟正常肝脏基本结构,提出仿肝板肝组织三维共培养技术的研究。通过该培养模式下对肝细胞功能进行测定,并和其他体外培养模式比较,以期得到能够提高体外肝细胞功能并适于生物人工肝应用的新的肝细胞培养模式。另外,这种仿肝板结构三维培养的肝组织也有望成为药物开发和检测的有效评价工具。研究方法和内容:我们利用微流控、水凝胶、软光刻等技术,采用人源性肝细胞、内皮细胞共培养,成功构建出仿肝板样组织结构。为研究仿肝板肝组织三维共培养对肝细胞功能的影响,我们设计了体外功能实验和动物救治实验。仿肝板组和混合无序三维共培养、“三明治”夹层共培养、普通平板共培养法相比较,对四组进行为期两周的形态观察、肝细胞活力测定、肝细胞功能检测(白蛋白、谷丙转氨酶、凝血因子等)。动物实验部分,对雄性昆明小鼠联合应用D-氨基半乳糖和脂多糖进行急性肝衰竭造模,仿肝板肝组织腹腔注射对小鼠进行救治,观察昆明小鼠生存率,同时进行小鼠血液生化指标检测,综合评估仿肝板肝组织对小鼠的救治效果。实验结果:体外实验中,仿肝板组和混合三维组大体形态上无明显差异;仿肝板组肝细胞活力高于混合三维组;仿肝板组在CYP1A2、凝血因子Ⅱ、睾酮代谢、安定代谢等指标方面明显优于另外三组。动物实验部分,仿肝板组救治效果明显,和对照组相比小鼠生存率明显提高,多数生化指标下降明显。实验结论:仿肝板肝组织三维共培养对肝细胞功能的影响表现在各个方面。在肝细胞营养物质代谢、蛋白合成分泌、药物代谢等方面,与“三明治”夹层共培养、普通平板共培养法相比较,具有明显的优势,值得进一步研究。仿肝板肝组织能够部分替代肝衰竭昆明小鼠的肝脏功能,且对小鼠血液生化指标有一定改善效果。本实验采用人源性细胞,为生物人工肝、药物开发与检测、细胞移植等提供了一定的参考价值。
[Abstract]:Background liver failure is caused by a variety of factors (such as viruses, alcohol, drugs, etc.), resulting in a large number of necrosis of liver cells, leading to serious dysfunction of liver function or decompensation, and then to clotting mechanism disorders, jaundice, hepatic encephalopathy, etc. Ascites are a group of clinical symptoms. At present, liver transplantation is an effective method to treat liver failure. However, organ transplantation in countries all over the world is facing a serious shortage of sources. The reconstruction of artificial liver and liver tissue engineering provide a new choice for the treatment of liver failure. Objective and significance: we simulate the basic structure of normal liver by combining liver tissue engineering with bioartificial liver technology. A three-dimensional co-culture technique for liver tissue imitating liver plate was proposed. The function of hepatocytes was measured in this culture model and compared with other culture models in vitro. In order to obtain a new hepatocyte culture model, which can improve the function of hepatocytes in vitro and be suitable for bioartificial liver. It is expected that the three-dimensional culture of liver tissue will also be an effective evaluation tool for drug development and detection. Methods and contents: we use microfluidic, hydrogel, soft lithography and other techniques to use human hepatocytes. In order to study the effect of three-dimensional co-culture of liver imitating tissue on the function of hepatocytes, endothelial cell co-culture was successfully constructed. In vitro functional experiment and animal rescue experiment were designed. The four groups were observed for two weeks, such as liver imitating plate group, mixed disordered three-dimensional co-culture, sandwich sandwich co-culture and common plate co-culture. Determination of hepatocyte activity, determination of hepatocyte function (albumin, alanine aminotransferase, coagulation factor, etc.) in animal experiment, the model of acute liver failure in male Kunming mice was established by combined use of D-galactose and lipopolysaccharide. The survival rate of Kunming mice was observed by intraperitoneal injection of liver imitating liver tissue. Meanwhile, the blood biochemical indexes of mice were detected, and the therapeutic effect of liver tissue on mice was comprehensively evaluated. There was no significant difference in gross morphology between the imitating liver plate group and the mixed three dimensional group, the hepatocyte activity in the imitating liver plate group was higher than that in the mixed three dimensional group, and the liver imitating plate group was in CYP1A2, coagulation factor 鈪，

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