MCP-1和MIP-2在新生鼠胆道闭锁肝内胆管损伤及肝纤维化中的作用

Mechanisms of bile duct injury and liver fibrosis induced by MCP-1 and MIP-2 in liver tissues of biliary atresia mice

目的建立轮状病毒（RRV）感染Balb／c新生鼠致胆道闭锁（biliaryatresia，BA）动物模型，并探讨单核细胞趋化蛋白-1（MCP-1）和巨噬细胞炎性蛋白-2（MIP-2）在BA新生鼠肝内胆管损伤及肝纤维化过程中的作用机制。方法采用腹腔注射猴MMUl8006轮状病毒毒株，诱导Balb／c新生鼠产生胆道闭锁作为实验组，腹腔注射相同剂量的病毒培养液（10％FBS-DMEM，10％胎牛血清达尔伯克改良伊格尔培养基）为对照组。在注射后4、7、14及21d各处死一批小鼠，取得胆管及肝组织，通过苏木精-伊红（HE）染色分析其肝外胆管及肝脏组织学改变，免疫组化法检测MCP-1和MIP-2在肝组织中的表达，Masson染色观察肝内纤维化程度。应用Image-ProPlus图像分析软件对MCP-1和MIP-2及肝纤维化程度进行半定量分析。结果实验组小鼠体重增长明显慢于对照组，且出现皮肤黄染等胆道梗阻症状；实验组小鼠肝胆系统出现急慢性炎症反应，且可见肝外胆管狭窄（7d）及闭锁（14d）；实验组小鼠肝内MCP-1和MIP-2的表达量在各时期内均明显高于对照组（21d时，实验组小鼠肝内MCP-1和MIP-2的阳性指数分别为24902．85±2420．38、6215．47±469．83，而对照组分别为5168．16±627．30、783．38±46．19，Pd0．001），并且两者成正相关（r=0．918，P〈0．001）；实验组小鼠肝实质内可见大量胶原蛋白沉积[21d时，实验组小鼠肝内胶原蛋白所占面积为（35．30±5．45）％，而对照组为（6．71±0．83）％，P〈O．001]，甚至纤维化，并且胶原蛋白沉积面积与MCP-1表达量成正相关（r=0．529，P=0．005），而对照组未见明显胶原蛋白沉积。结论BA是一种由病毒感染诱发、多种炎症因子介导的免疫炎性疾病，MCP-1以及MIP-2的高表达可能在诱导BA新生鼠胆管损伤及肝纤维化方面起重要作用。

Objective To establish a model of biliary atresia （BA） by infecting neonatal mice with rotavirus （RRV） and explore the mechanisms of intra-hepatic bile duct injury and liver fibrosis under a high expression of MCP-1 and MIP-2 in BA murine liver. Methods RRV was injected intraperitoneally to induce BA of neonatal mice as experimental group while 10% fetal bovine serum-Dulbecco＇s modification of Eagle＇s medium （FBS-DMEM） was injected as control group. The neonatal mice were sacrificed at Days 4, 7, 14 and 21 and their extra-hepatic bile ducts and liver tissues were harvested. The histological changes of extra-hepatic bile ducts and liver tissues were analyzed by hematoxylin-eosin staining. The expressions of MCP-1 and MIP-2 in BA mice livers were detected by immunohistochemistry and fibrosis by Masson staining. And Image-Pro Plus software was used to measure the positive indices of MCP-I and MIP-2 and the fibrosis level of liver tissues. Results The gain weights of BA mice were significantly slower than those of control group. And the BA mice had typical symptom of jaundice. HE staining showed plenty of cellular infiltrates in both bile ducts and liver tissues of BA group, partially narrowed bile ducts were observed at Day 7 and obliterated bile ducts at Day 14. The expressions of MCP-1 and MIP-2 were significantly higher than those of control group. The positive indices of MCP-1 and MIP-2 in BA mice livers were 5168. 16 ±627. 30 and 783.38 ± 46. 19 respectively at Day 21 days versus control group of 5168. 16 ± 627. 30 and 783.38 ± 46. 19 （P〈0. 001. There was a strong positive correlation with each other （r = 0. 918, P〈 0. 001）. A large number of collagen deposition and even fibrosis were found in liver tissues of BA group. Collagen deposition in BA mice livers were （35. 3 ± 5.45） % at Day 21 versus control group of （6. 71 ± 0. 83）% （P〈0. 001）. And there was a positive correlation between collagen deposition and MCP-1 expression （r= 0. 529, P = 0. 005） and it was not observed in control group. Conclusions BA is an inflammatory disease probably mediated by a variety of inflammatory cytokines induced by virus infection. A high expression of MCP-1 and MIP-2 may play an important role in promoting inflammatory cell infiltration and collagen deposition leading to bile duct injury and liver fibrosis of BA mice.