Lu Xu-Feng, Zhou Yong-Jie, Zhang Lei, Ji Hong-Jie, Li Li, Shi Yu-Jun, Bu Hong
Xu-Feng Lu, Yong-Jie Zhou, Lei Zhang, Hong-Jie Ji, Li Li, Yu-Jun Shi, Hong Bu, Laboratory of Pathology, West China Hospital, Sichuan University, Chengdu 610041, Sichuan Province, China.
World J Gastroenterol. 2015 Jun 7;21(21):6591-603. doi: 10.3748/wjg.v21.i21.6591.
To investigate the continuous hepatic histopathological processes which occur in response to the loss of Dicer1.
We generated a hepatocyte-selective Dicer1 knockout mouse and observed the gradual hepatic histopathological changes in the mutant liver. Immunohistochemistry and Western blotting were performed to detect Dicer1 expression. We performed hematoxylin and eosin staining, Periodic acid-Schiff staining, Oil Red O staining, and Masson's trichrome staining to detect histological changes in Dicer1-deficient livers. Ki67 immunohistochemistry, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, and Western blotting were used to determine hepatocyte proliferation and apoptosis. Serum biochemistry, cytokine assays, and flow cytometric analysis were performed to quantity liver necrosis and inflammation. Fibrogenic markers were determined by Western blotting and qPCR. CK19, CD133, and OV6 immunofluorescence were used to observe liver progenitor cells. Immunofluorescence and qPCR were performed to reveal embryonic gene expression. We also performed histological staining and Western blotting to analyze hepatocellular carcinoma (HCC) development.
Dicer1 inactivation resulted in significant architecture disorganization and metabolism disruption in the liver. Dicer1 disruption impaired hepatocyte survival and resulted in profound cell apoptosis and continuous necrosis. In contrast to previous reports, the mutant liver exhibited chronic inflammation and progressive fibrosis, and could not be repopulated by Dicer1-positive cells. In addition, extensive activation of hepatic progenitor cells was observed. Primary HCC was observed as early as 4 mo after birth.
Hepatic loss of Dicer1 results in complex chronic pathological processes, including hepatocyte death, inflammatory infiltration, chronic fibrosis, compensatory proliferation, progenitor activation, and spontaneous hepatocarcinogenesis.
研究因Dicer1缺失而发生的持续性肝脏组织病理学变化过程。
我们构建了肝细胞选择性Dicer1基因敲除小鼠，并观察突变肝脏中逐渐出现的组织病理学变化。采用免疫组织化学和蛋白质免疫印迹法检测Dicer1表达。我们进行苏木精-伊红染色、过碘酸-希夫染色、油红O染色和Masson三色染色，以检测Dicer1缺陷肝脏中的组织学变化。采用Ki67免疫组织化学、末端脱氧核苷酸转移酶介导的dUTP缺口末端标记法和蛋白质免疫印迹法来确定肝细胞增殖和凋亡情况。进行血清生化、细胞因子检测和流式细胞术分析，以量化肝脏坏死和炎症情况。通过蛋白质免疫印迹法和定量聚合酶链反应测定纤维化标志物。采用细胞角蛋白19、CD133和OV6免疫荧光法观察肝脏祖细胞。采用免疫荧光法和定量聚合酶链反应揭示胚胎基因表达情况。我们还进行组织学染色和蛋白质免疫印迹法分析肝细胞癌（HCC）的发生情况。
Dicer1失活导致肝脏结构显著紊乱和代谢破坏。Dicer1缺失损害肝细胞存活，导致严重的细胞凋亡和持续性坏死。与之前的报道不同，突变肝脏表现出慢性炎症和进行性纤维化，且不能被Dicer1阳性细胞重新填充。此外，观察到肝脏祖细胞广泛激活。出生后4个月就观察到原发性HCC。
肝脏中Dicer1缺失导致复杂的慢性病理过程，包括肝细胞死亡、炎症浸润、慢性纤维化、代偿性增殖、祖细胞激活和自发性肝癌发生。
