本文選題：生物人工肝　切入點(diǎn)：海藻酸　出處：《南方醫(yī)科大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：研究背景肝衰竭是指受到多種因素(如病毒、酒精、藥物等)造成肝細(xì)胞大量壞死,導(dǎo)致肝臟功能發(fā)生嚴(yán)重障礙或失代償,進(jìn)而出現(xiàn)以凝血機(jī)制障礙、黃疸、肝性腦病、腹水等為主要表現(xiàn)的一組臨床癥候群。目前肝移植是治療肝衰竭的有效方法,然而世界各國(guó)的器官移植面臨著嚴(yán)重的來(lái)源短缺問(wèn)題。為此,以實(shí)現(xiàn)肝臟功能修復(fù)、替代、重建為目標(biāo)的人工肝技術(shù)和肝組織工程為肝衰竭治療提供了新的選擇。研究目的和意義:我們通過(guò)結(jié)合肝組織工程與生物人工肝技術(shù),模擬正常肝臟基本結(jié)構(gòu),提出仿肝板肝組織三維共培養(yǎng)技術(shù)的研究。通過(guò)該培養(yǎng)模式下對(duì)肝細(xì)胞功能進(jìn)行測(cè)定,并和其他體外培養(yǎng)模式比較,以期得到能夠提高體外肝細(xì)胞功能并適于生物人工肝應(yīng)用的新的肝細(xì)胞培養(yǎng)模式。另外,這種仿肝板結(jié)構(gòu)三維培養(yǎng)的肝組織也有望成為藥物開(kāi)發(fā)和檢測(cè)的有效評(píng)價(jià)工具。研究方法和內(nèi)容:我們利用微流控、水凝膠、軟光刻等技術(shù),采用人源性肝細(xì)胞、內(nèi)皮細(xì)胞共培養(yǎng),成功構(gòu)建出仿肝板樣組織結(jié)構(gòu)。為研究仿肝板肝組織三維共培養(yǎng)對(duì)肝細(xì)胞功能的影響,我們?cè)O(shè)計(jì)了體外功能實(shí)驗(yàn)和動(dòng)物救治實(shí)驗(yàn)。仿肝板組和混合無(wú)序三維共培養(yǎng)、“三明治”夾層共培養(yǎng)、普通平板共培養(yǎng)法相比較,對(duì)四組進(jìn)行為期兩周的形態(tài)觀察、肝細(xì)胞活力測(cè)定、肝細(xì)胞功能檢測(cè)(白蛋白、谷丙轉(zhuǎn)氨酶、凝血因子等)。動(dòng)物實(shí)驗(yàn)部分,對(duì)雄性昆明小鼠聯(lián)合應(yīng)用D-氨基半乳糖和脂多糖進(jìn)行急性肝衰竭造模,仿肝板肝組織腹腔注射對(duì)小鼠進(jìn)行救治,觀察昆明小鼠生存率,同時(shí)進(jìn)行小鼠血液生化指標(biāo)檢測(cè),綜合評(píng)估仿肝板肝組織對(duì)小鼠的救治效果。實(shí)驗(yàn)結(jié)果:體外實(shí)驗(yàn)中,仿肝板組和混合三維組大體形態(tài)上無(wú)明顯差異;仿肝板組肝細(xì)胞活力高于混合三維組;仿肝板組在CYP1A2、凝血因子Ⅱ、睪酮代謝、安定代謝等指標(biāo)方面明顯優(yōu)于另外三組。動(dòng)物實(shí)驗(yàn)部分,仿肝板組救治效果明顯,和對(duì)照組相比小鼠生存率明顯提高,多數(shù)生化指標(biāo)下降明顯。實(shí)驗(yàn)結(jié)論:仿肝板肝組織三維共培養(yǎng)對(duì)肝細(xì)胞功能的影響表現(xiàn)在各個(gè)方面。在肝細(xì)胞營(yíng)養(yǎng)物質(zhì)代謝、蛋白合成分泌、藥物代謝等方面,與“三明治”夾層共培養(yǎng)、普通平板共培養(yǎng)法相比較,具有明顯的優(yōu)勢(shì),值得進(jìn)一步研究。仿肝板肝組織能夠部分替代肝衰竭昆明小鼠的肝臟功能,且對(duì)小鼠血液生化指標(biāo)有一定改善效果。本實(shí)驗(yàn)采用人源性細(xì)胞,為生物人工肝、藥物開(kāi)發(fā)與檢測(cè)、細(xì)胞移植等提供了一定的參考價(jià)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，

本文編號(hào)：1575710