C1型尼曼-匹克病小鼠肝脏功能及病理变化

杨记超 宋莹 刘达 仝曼 张阳 管丽红 乔梁* 林俊堂*

doi:10.16098/j.issn.0529-1356.2018.06.009

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解剖学报 ›› 2018, Vol. 49 ›› Issue (6) : 745-751. DOI: 10.16098/j.issn.0529-1356.2018.06.009

组织学胚胎学发育生物学

C1型尼曼-匹克病小鼠肝脏功能及病理变化

杨记超^1,5 宋莹^2,5 刘达²仝曼⁴ 张阳⁴管丽红^2,5 乔梁^2,5* 林俊堂^3,5*

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Liver function and pathological changes in Niemann-Pick disease type C1 mice

YANG Ji-chao^1,5 SONG Ying ^2,5 LIU Da²TONG Man⁴ZHANG Yang⁴ GUAN Li-hong^{2, 5} QIAO Liang ^{2, 5*} LIN Jun-tang ^{3, 5*}

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摘要

目的探讨晚期（P60）Npc1^-/-小鼠肝脏功能和病理变化，为C1型尼曼-匹克病（NPC1）患者肝脏的病理发生及临床治疗提供实验依据。方法小鼠称重后，眼内眦取血检测血清中乳酸脱氢酶（LDH）、丙氨酸氨基转移酶（ALT）和天冬氨酸氨基转移酶（AST）的活性变化，评价Npc1^-/-小鼠的肝功能变化；取肝组织进行石蜡和冷冻切片，通过HE染色和油红O染色评估肝脏组织的形态变化和脂肪储存情况，以及Masson染色评估肝脏组织胶原沉积情况；Real-time PCR和Western blotting分别检测肝脏组织促炎症因子，白细胞介素（IL）-1β、IL-6和肿瘤坏死因子（TNF）-α的mRNA和蛋白水平表达情况；TUNEL染色评估肝脏组织凋亡情况。结果与Npc1^+/+小鼠相比，Npc1-/-小鼠的体重及肝脏系数显著降低（P<0.001）；LDH、ALT及AST活性显著升高（P<0.001）；HE染色发现，Npc1^-/-小鼠肝脏组织形态改变明显，出现大量泡沫细胞；油红O染色显示，Npc1^-/-小鼠肝脏脂肪含量显著降低；Real-time PCR显示，Npc1^-/-小鼠肝脏组织IL-1β、IL-6和TNF-α表达均显著升高（P<0.01，P<0.05和P<0.001），同时，Western blotting也显示，3个因子的蛋白表达均显著升高（P<0.05，P<0.05和P<0.01），证实Npc1-/-小鼠肝脏晚期发生炎症反应；Masson染色未发现Npc1^-/-小鼠肝脏纤维化的发生；TUNEL染色显示，Npc1^-/-小鼠肝脏组织凋亡细胞数量增加。结论 Npc1基因突变导致肝脏组织形态发生改变，大量的巨噬细胞聚集引发炎症反应，而炎症反应的发生可能是引起肝脏细胞凋亡和肝功能受损的重要途径之一。

Abstract

Objective To explore the function and pathology of liver in late Npc1-/-mice (P60) and provide theoretical basis for the pathological and clinical treatment of Niemann-Pick disease type C1 (NPC1). Methods Weighing the mice, the activity of lactate dehydrogenase (LDH), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum of eye canthus were analyzed to evaluate liver function of Npc1-/- mouse. The liver tissues were treated with paraffin and then frozen sections and stained HE staining and oil red O staining was used to observe the morphological changes and fat storage in liver tissue. The collagen deposition in liver tissue was evaluated by Masson staining. The Real-time PCR and Western blotting were used to detect the expression of proinflammatory factors interleukin（IL）-1β, IL-6 and tumer necrosis factor(TNF)-α in liver tissue. The apoptosis of liver tissue was observed by TUNEL staining. Results Compared with the Npc1+/+ mouse, the body weight and liver coefficient of Npc1-/- mouse were decreased significantly (P<0.001), and the activity of LDH, ALT and AST were increased significantly (P<0.001). HE staining showed that morphological changes of the liver were obvious in Npc1-/- mice, and a large number of foam cells appeared. Oil red O staining showed a significant decrease in the positive rate of liver fat cells in Npc1-/- mice. Real-time PCR showed that the expression of IL-1β, IL-6 and TNF-α in the liver of Npc1-/- mice increased significantly (P<0.01,P<0.05 andP<0.001). Western blotting showed that the expression of pro-inflammatory factors IL-1β, IL-6 and TNF-α protein increased (P<0.05, P<0.05 and P<0.01); Inflammatory reaction in the late liver; Masson staining showed no obvious collagen deposition in the liver of Npc1-/- mice. TUNEL staining showed that the number of apoptosis cells increased in the liver of Npc1-/- mice. Conclusion The mutation of Npc1 gene leads to the morphological changes of the liver and the amounts of macrophage aggregation. These increases in macrophage aggregation may cause severe inflammatory reactions in the liver. However, the occurrence of inflammatory reactions in liver may play a crucial role in promoting liver cell apoptosis and impaired liver function.

导出引用

杨记超宋莹刘达仝曼张阳管丽红乔梁林俊堂. C1型尼曼-匹克病小鼠肝脏功能及病理变化[J]. 解剖学报. 2018, 49(6): 745-751 https://doi.org/10.16098/j.issn.0529-1356.2018.06.009

YANG Ji-chao SONG Ying LIU Da TONG Man ZHANG Yang GUAN Li-hong QIAO Liang LIN Jun-tang. Liver function and pathological changes in Niemann-Pick disease type C1 mice[J]. Acta Anatomica Sinica. 2018, 49(6): 745-751 https://doi.org/10.16098/j.issn.0529-1356.2018.06.009

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