小鼠酒精性肝纤维化模型的建立及酒精性肝病的临床分析

发布时间：2018-04-02 04:19

  本文选题：肝疾病/酒精性　切入点：肝纤维化　出处：《第一军医大学》2007年硕士论文

【摘要】： 酒精性肝病(Alcoholic liver disease，ALD)是由于长期大量饮酒导致的肝脏疾病。根据中华医学会肝脏病学分会提出的ALD病理诊断标准，分轻型酒精性肝病(mild alcoholic injury，MAI)、酒精性脂肪肝(alcoholic fatty liver，AFL)、酒精性肝炎(alcoholic hepatic，AH)、酒精性肝纤维化(alcoholic hepatic fibrosis，AHF)、酒精性肝硬化(alcoholic cirrhosis，AC)5种类型。ALD是西方国家导致肝硬化的最主要的病因，也是十大常见死因之一。近年来随着人们生活方式的改变，我国由酒精所至肝损害的发病率亦呈逐年上升趋势，成为继病毒性肝炎后导致肝损害的第二大病因。肝纤维化是肝细胞发生坏死或炎症刺激时，肝内纤维结缔组织异常增生的病理过程，进一步发展可引起肝小叶结构改建、假小叶及结节形成，即肝硬化。国内外学者对肝纤维化的可逆性已无异议，故酒精性肝纤维化对酒精性肝硬化的出现与否及疾病的预后起着决定性的作用，其演变和逆转近年来倍受关注。为深入研究酒精性肝病的发病机制，筛选有效预防、治疗本病的药物及方法，建立与人类酒精性肝损伤病变过程相似的动物模型具有积极的现实意义。 目的 本文第一部分旨在探索建立较为理想的小鼠酒精性肝纤维化模型，为研究骨髓衍生肝干细胞对酒精性肝损伤的修复提供动物模型。本文第二部分对我院近5年来收治的105例酒精性肝病患者进行临床资料分析，探讨酒精性肝病的发病特点、临床表现及预后，以提高对酒精性肝病的认识和重视，为早期发现、早期诊断、早期治疗酒精性肝病提供依据。 方法和结果 1、小鼠酒精性肝纤维化模型的建立 Balb／c雌性小鼠100只，SPF级，5～6周龄，体重15～18g，适应环境一周后，随机分为造模组92只，对照组8只。造模组给予54度白酒每天灌胃2次共16周，同时以10％(v／v)白酒为唯一饮料。第1周～第3周末灌胃量为每次8mL／kg，第4周～第7周末灌胃量为每次10 mL／kg，第8周～第16周末灌胃量为每次12 mL／kg。对照组给予等量蒸馏水灌胃，饮用自来水。实验期间均予全价营养颗粒饲料喂养。造模组于白酒灌胃的第4，8，12，16周末分别随机抽样选取6只小鼠，眼球采血后，颈椎脱臼处死，处死前禁食12h。处死后立即取出肝脏，称重后以10％的甲醛固定，脱水后石腊包埋切片，苏木精—伊红染色(HE)和苦味酸—酸性品红染色(VG)，光镜观察病理变化。血标本用于测定生化指标。对照组小鼠于第16周末处死，处理同造模组。实验过程中观察小鼠的精神状态、活动情况、皮毛光泽度、食欲等。肝脏标本观察肝脏的大小、外形、色泽、质地、切面情况。取血清进行生化指标的检测，包括丙氨酸氨基转移酶(ALT)，天冬氨酸氨基转移酶(AST)，甘油三脂(TG)，总蛋白(TP)，白蛋白(ALB)。病理学观察：肝组织常规石腊包埋切片，HE染色和VG染色，用于观察肝脏病理学改变(肝细胞变性、坏死、炎性细胞浸润、胶原纤维增生等)，结果如下。 1．1实验动物数量分析：实验过程中造模组共23只小鼠死亡，多为急性和亚急性死亡。原因主要有：窒息、消化道穿孔、急性胃扩张和酒精中毒等。对照组全部成活。 1．2精神变化：灌胃初期，造模组小鼠出现精神萎靡、嗜睡、反应迟钝、活动减少、食欲减退等现象，实验开始4周后上述症状减轻，但皮毛光泽度差，体重增长缓慢。对照组小鼠则皮毛光泽、体态活泼、食欲正常，无嗜睡现象。 1．3肝脏标本的外观：对照组小鼠肝脏表面光滑细润、色红、边缘锐利、质地中等。相比之下，造模组小鼠肝脏色泽较正常肝脏暗淡，表面充血。 1．4组织的病理学改变：对照组HE染色标本见肝细胞以中央静脉为中心呈放射状排列，肝小叶轮廓清晰，肝索排列整齐。造模组4周时可见轻度脂肪变性，肝细胞浊肿，胞浆中出现大小不等的脂滴；8周时，细胞索紊乱，，胞浆肿大疏松化，其内细胞核固缩，可见肝细胞广泛性的空泡样变性，脂肪变性较4周模型组小鼠增多；12周时，肝细胞点状及灶状坏死较多见，汇管区有炎性细胞浸润，一些坏死区可见纤维细胞增生；16周时，炎症坏死和纤维化更加明显，汇管区有明显红色胶原纤维增生，并向周围肝小叶内延伸。 1．5血清生化指标：各周造模组小鼠血清中白蛋白含量与正常对照组比较差异显著(F=6.490，P=0.001)；各周模型组相比，血清白蛋白含量没有显著差异(F=0.620，P=0.610)。造模组血清总蛋白的含量与对照组相比，在造模的第4，8，12，16周时没有明显改变，差异不显著(F=1.217，P=0.327)；造模各时间点间两两比较，总蛋白含量也没有显著性差异(F=0.965，P=0.429)。各周造模组小鼠血清中AST、ALT、TG含量和AST／ALT与对照组相比，除4周模型组AST／ALT、TG与正常对照组相比无显著性差异外(P=0.083；P=0.318)，其余均有显著性差异(F=10.281，P＜0.001；F=8.610，P=＜0.001；F=3.605，P=0.018；F=4.490，P=0.007)。 2、酒精性肝病105例临床分析 依据患者的饮酒史、临床表现、肝功能指标及影像学、病理学检查的结果，按照中华医学会肝病学分会脂肪肝和酒精性肝病学组2006年2月修订的酒精性肝病诊疗指南上的酒精性肝病临床诊断标准，将我院近5年来收治的105例酒精性肝病患者分为酒精性脂肪肝组(AFL)、酒精性肝炎组(AH)、酒精性肝硬化组(AC)，对其数据进行统计学分析，并做出临床分析和讨论，其结果如下。 2．1 ALD临床表现与其他原因所致肝病类似，无特异性，常见症状为乏力、纳差、发热、黄疸、腹胀、呕血、黑便。 2．2 ALD患者男性为主，日饮酒量45～600g，平均年限21年。本资料中ALD患者年龄在30～60之间的病例数最多(89例)。酒精性肝硬化67例，占63.8％。AC组日饮酒量同AFL组、AH组相比，差异显著(P=0.001，P=0.013)；AC组饮酒时间(24.13±7.84)年同AFL组(16.21±12.43)年、AH组(16.00±10.09)年相比也均有显著性差异(P=0.001，P=0.001)。 2．3 ALD患者的血清酶学水平有不同程度的异常，血清中ALT、AST、GGT的水平以AH组为最高，同AFL组相比，差异显著(P＜0.001)；AH组患者血清中AST、ALT、GGT的水平同AC组患者相比，差异显著(P＜0.001；P＜0.001；P=0.047)。各组间AST／ALT相比无显著性差异(F=0.801，P=0.451)。血清ALB以AC组为最低，分别与AH组、AFL组相比均有显著性差异(P=0.003；P＜0.001)。TBIL以AC组最高，3组间差异有显著性意义(P＜0.001)。ALD患者死亡的主要原因是肝硬化的晚期并发症。 结论 1、应用白酒灌胃法成功的复制了小鼠酒精性肝纤维化模型，并在16周内观察到酒精性肝病的一些病理表现，如脂肪变性、炎症改变、胶原纤维的增生。随着酒精刺激时间的延长，肝脏的损伤程度加重。 2、此种建模方法接近国人的饮酒习惯、方法简便易行、费用低，可为研究骨髓衍生肝干细胞对酒精性肝损伤的修复提供较理想的动物模型。 3、本研究中ALD患者高峰年龄在30～60之间，饮酒量、饮酒时间与酒精性肝病的发病关系密切。 4、因酒精性肝病临床表现无特异性，对于有饮酒嗜好的病人，应定期对AST、AST／ALT，GGT，TBIL等生化指标监测并结合影像学检查，从而争取早期诊断、早期治疗、改善预后。
[Abstract]:Alcohol liver disease ( ALD ) is a major cause of liver injury due to chronic alcoholic liver disease ( ALD ) .






Purpose






The first part of this paper is to explore the establishment of an ideal model of alcoholic liver fibrosis in mice , and to provide an animal model for the study of the repair of alcoholic liver injury by bone marrow derived liver stem cells . The second part of this paper deals with the clinical data of 105 patients with alcoholic liver disease treated in our hospital in recent 5 years , and discusses the pathogenesis , clinical manifestation and prognosis of alcoholic liver disease , so as to improve the awareness and attention of alcoholic liver disease . It provides the basis for early detection , early diagnosis and early treatment of alcoholic liver disease .






Methods and Results






1 . Establishment of model of alcoholic liver fibrosis in mice






Blood samples were used to measure biochemical indexes . The rats were randomly divided into two groups at 8 mL / kg every 8 mL / kg and 8 weeks to 16 weeks .






1.1 The number of experimental animals showed that 23 mice died during the experiment , mostly acute and subacute death . The main reasons were asphyxia , perforation of digestive tract , acute gastric dilatation and alcoholism . All the control groups were alive .






P < 0.001 ;






1.3 Appearance of the liver specimen : the liver surface of the control group was smooth and smooth , the color was red , the edge was sharp , and the texture was middle . In contrast , the liver of the model mice had a normal liver color , and the surface was filled with blood .






1.4 The pathological changes of the liver tissues were as follows : the control group HE staining samples showed that the liver cells were radially arranged in the central vein , the hepatic lobule was clear and the liver was arranged orderly .
At 8 weeks , the cell line was disturbed , the cytoplasm was loose , the cells in its inner cells were pyknosis , the vacuolar degeneration of hepatocytes was seen , and the number of mice with fatty degeneration was more than 4 weeks .
At 12 weeks , the hepatocyte punctate and focal necrosis were more common , inflammatory cell infiltration in the manifold area , and the proliferation of some necrotic areas .
At 16 weeks , inflammatory necrosis and fibrosis were more evident , and the manifold area had a marked red collagen fiber hyperplasia and extended to the surrounding liver leaflets .






1.5 Serum biochemical indexes : The serum albumin content of every peripheral group was significantly different from that of the normal control group ( F = 6.490 , P = 0.001 ) .
There was no significant difference in serum albumin content ( F = 0.620 , P = 0.610 ) . Compared with the control group , there was no significant difference between the serum total protein content and the control group ( F = 1.217 , P = 0.327 ) .
There was no significant difference ( F = 0.965 , P = 0.429 ) . Compared with control group , AST , ALT , TG and AST / ALT were not significantly different from normal control group ( P = 0.083 ) .
There was significant difference ( F = 10.281 , P < 0.001 ) .
F=8.610,P=锛

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