Differential expression of hepatic fibrosis mediators in sick and spontaneously recovered mice with experimental biliary atresia.
Differential expression of hepatic fibrosis mediators in sick and spontaneously recovered mice with experimental biliary atresia.
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患有实验性胆道闭锁的患病和自然康复小鼠中肝纤维化介质的差异表达。
DOI：
10.1016/j.jss.2009.10.038
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发表时间：
2010-04
期刊：
影响因子：
--
通讯作者：
Greco MA
中科院分区：
文献类型：
--
作者：
Nadler EP;Li X;Onyedika E;Greco MA
导致肝硬化的肝纤维化是胆道闭锁（BA）患者的主要发病率。这种纤维化是由于细胞外基质（ECM）分解和沉积的不平衡造成的。我们之前已经证明，在我们的 BA 动物模型中，到第 14 天时，ECM 分解抑制剂的 mRNA 表达增加，而 ECM 沉积介质的表达没有增加。然而，此时仅观察到轻度肝纤维化。我们假设一旦出现更明显的纤维化，这些蛋白质的表达模式可能会发生变化，并在模型中添加复苏以提高生存率。有趣的是，我们发现一些小鼠在随后的时间点通过复苏自然恢复，因此比较了患病和康复小鼠中 ECM 分解和沉积抑制剂的表达以确定差异。新生Balb/c小鼠在出生24h后腹腔注射1.0×106荧光形成单位的恒河猴轮状病毒。每天监测小鼠的体重增加、黄疸、无胆汁粪便和胆红素尿的发生情况。从第 7 天开始，每天向每只小鼠皮下注射 50 μL/g 5% 葡萄糖的生理盐水，直至处死。第 14 天后存活的小鼠在盐水或 RRV 感染后第 21 天被处死。然后在注射后第21天收获肝脏用于组织学和免疫组织化学分析。使用定量实时 PCR 评估已知纤维化介质的 RNA 表达。使用ELISA评估蛋白质表达。使用学生 t 检验比较权重和正态分布数据。使用费舍尔精确检验比较组织学结果。通过 Mann-Whitney U 检验进行基因表达和偏态数据的比较。任何小于 0.05 的 P 值均具有统计学显着性。在我们的模型中，每日复苏导致第 21 天的存活率为 35% (24/68)。康复的小鼠在第 14 天（6.15 ± 1.16 对比 4.94 ± 0.82，P = 0.02）和 21 天（7.31 ± 1.41 对比 4.14 ± 0.53，P < 0.001）显着重于仍然患病的小鼠，尽管事实上各组之间在第 7 天的体重没有差异（4.29 ± 0.90 与 3.89 ± 0.81，P = 0.32）。我们发现，在第 21 天表现出胆道闭锁临床症状的所有 (10/10) 动物均具有中度或重度组织学发现，而只有一只 (1/9) 康复动物在处死时出现肝脏异常（与患病组相比，P < 0.001）。我们还发现，与康复小鼠相比，患病小鼠的 TIMP-1（31.9 对比 9.1，P = 0.041）、TIMP-4（88.1 对比 1.8，P = 0.022）和 MMP-7（51.8 对比 11.9，P = 0.006）的 mRNA 表达中值倍数增加具有统计学意义。 PAI-1 mRNA 表达有下降趋势，但未达到统计学显着性（中位数 27.7 对比 2.19，P = 0.066）。在患病组中还发现 TIMP-1 和 PAI-1 蛋白表达增加。 RRV感染后21天，患病组和康复组中纤维状胶原、纤连蛋白-1、结缔组织生长因子、snail-1、TIMP-2和-3、MMP-2和MMP-9的mRNA表达没有差异，并且没有从基线基因表达升高。在 BA 动物模型中添加复苏后，一些小鼠自发康复，而另一些小鼠则进展为更严重的肝纤维化。肝纤维化小鼠的 TIMP-1、TIMP-4 和 MMP-7 mRNA 表达持续增加，第 21 天 PAI-1 mRNA 表达有增加的趋势。患病小鼠中 TIMP-1 和 PAI-1 的蛋白质水平也增加。康复的小鼠表现出轻度或无肝实质疾病，并且测试的纤维化介质的 mRNA 表达模式正常。两组中均未发现 ECM 沉积介质的 mRNA 表达增加。这些数据进一步支持这样的观点：仅抑制 ECM 分解就足以诱导肝纤维化。调节这一过程可能是预防 BA 患者肝损伤的假定目标。
Hepatic fibrosis leading to cirrhosis is the major morbidity in patients with biliary atresia (BA). This fibrosis is due to an imbalance in extracellular matrix (ECM) breakdown and deposition. We have previously demonstrated increased mRNA expression for inhibitors of ECM breakdown without increased expression for mediators of ECM deposition in our animal model of BA by d 14. However, only a mild degree of hepatic fibrosis was seen at this time. We hypothesized that expression patterns for these proteins may change once more significant fibrosis had been established, and added resuscitation to the model to improve survival. Interestingly, we found that some mice spontaneously recovered at later time points with resuscitation, and thus compared expression for inhibitors of ECM breakdown and deposition in sick and recovered mice to determine the differences. Newborn Balb/c mice received an intraperitoneal injection 1.0 × 106 fluorescence forming units of rhesus rotavirus 24h after birth. Mice were monitored daily for weight gain, development of jaundice, acholic stools, and bilirubinuria. Fifty μL/g of 5% dextrose in normal saline were subcutaneously injected daily to each mouse starting on d 7 until sacrifice. Mice that survived past d 14 were sacrificed at d 21 after saline or RRV infection. Livers were then harvested post-injection d 21 for histologic and immunohistochemical analysis. RNA expression of known mediators of fibrosis was evaluated using quantitative real-time PCR. Protein expression was assessed using ELISA. Weights and normally distributed data were compared using Student's t test. Histologic findings were compared using Fisher's exact test. Comparisons of gene expression and skewed data were performed by the Mann-Whitney U test. Statistical significance was assigned to any P value less than 0.05. Daily resuscitation resulted in a 35% (24/68) survival rate to d 21 in our model. Mice that recovered were significantly heavier than those that remained ill on d 14 (6.15 ± 1.16 versus 4.94 ± 0.82, P = 0.02) and 21 (7.31 ± 1.41 versus 4.14 ± 0.53, P < 0.001) despite the fact that there was no difference between the groups with respect to weight on d 7 (4.29 ± 0.90 versus 3.89 ± 0.81, P = 0.32). We found that all (10/10) animals that displayed clinical signs of biliary atresia on d 21 had moderate or severe histologic findings, while only one (1/9) of the recovered animals had liver abnormalities at sacrifice (P < 0.001 versus sick group). We also found that the sick mice had statistically significant median fold-increases of mRNA expression for TIMP-1 (31.9 versus 9.1, P = 0.041), TIMP-4 (88.1 versus 1.8, P = 0.022), and MMP-7 (51.8 versus 11.9, P = 0.006) compared with those that recovered. There was a trend toward decreased mRNA expression for PAI-1, which did not reach statistical significance (median 27.7 versus 2.19, P = 0.066). Increased protein expression for TIMP-1 and PAI-1 were also found in the sick group. The mRNA expression for the fibrillar collagens, fibronectin-1, connective tissue growth factor, snail-1, TIMP-2 and -3, and MMP-2 and MMP-9 was not different in the sick and recovered groups 21 d after RRV infection, and was not elevated from baseline gene expression. With resuscitation added to the animal model of BA, some mice spontaneously recover while others progress to more significant hepatic fibrosis. Mice with hepatic fibrosis have a continued increase in mRNA expression of TIMP-1, TIMP-4, and MMP-7, with a trend toward increased mRNA expression of PAI-1 on d 21. Protein levels for TIMP-1 and PAI-1 were also increased in the sick mice. Recovered mice display mild to no hepatic parenchymal disease and a normal pattern of mRNA expression for the mediators of fibrosis tested. No increase in mRNA expression for the mediators of ECM deposition was found in either group. These data further support the notion that inhibition of ECM breakdown alone is sufficient to induce hepatic fibrosis. Modulation of this process may be a putative target for preventing liver injury in patients with BA.
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影响因子：
2.4
作者：
Lee, SY;Chuang, JH;Chen, CL
通讯作者：
Chen, CL
影响因子：
2.4
作者：
Petersen, C;Biermanns, D;Mildenberger, H
通讯作者：
Mildenberger, H
DOI：
10.1073/pnas.96.22.12719
发表时间：
1999-10-26
影响因子：
11.1
作者：
George, J;Roulot, D;Bissell, DM
通讯作者：
Bissell, DM
影响因子：
13.5
作者：
Wang, HT;Vohra, BPS;Heuckeroth, RO
通讯作者：
Heuckeroth, RO
影响因子：
4.6
作者：
Elias GJ;Ioannis M;Theodora P;Dimitrios PP;Despoina P;Kostantinos V;Charalampos K;Vassilios V;Petros SP
通讯作者：
Petros SP