Abstract: Objective　To investigate the capillarization of liver sinusoidal endothelial cells (LSECs) and its association with hepatic fibrosis during the development of alveolar echinococcosis, so as to provide the basis for unraveling the mechanisms underlying the role of LSEC in the development and prognosis of hepatic injuries and hepatic fibrosis caused by alveolar echinococcosis. Methods　Forty C57BL/6 mice at ages of 6 to 8 weeks were randomly divided into a control group and 1⁃, 2⁃ and 4⁃week infection groups, of 10 mice in each group. Each mouse in the infection groups was intraperitoneally injected with 2 000 Echinococcus multilocularis protoscoleces, while each mouse in the control group was given an equal volume of phosphate⁃buffered saline using the same method. All mice were sacrificed 1, 2 and 4 weeks post⁃infection and mouse livers were collected. The pathological changes of livers were observed using hematoxylin⁃eosin (HE) staining, and hepatic fibrosis was evaluated through semi⁃quantitative analysis of Masson’s trichrome staining⁃positive areas. The activation of hepatic stellate cells (HSCs) and extracellular matrix (ECM) deposition were examined using immunohistochemical staining of α⁃smooth muscle actin (α⁃SMA) and collagen type I alpha 1 (COL1A1), and the fenestrations on the surface of LSECs were observed using scanning electron microscopy. Primary LSECs were isolated from mouse livers, and the mRNA expression of LSEC marker genes Stabilin⁃1, Stabilin⁃2, Ehd3, CD209b, GATA4 and Maf was quantified using real⁃time fluorescence quantitative PCR (qPCR) assay. Results　Destruction of local liver lobular structure was observed in mice 2 weeks post⁃infection with E. multilocularis protoscoleces, and hydatid cysts, which were surrounded by granulomatous tissues, were found in mouse livers 4 weeks post⁃infection. Semi⁃quantitative analysis of Masson’s trichrome staining showed a significant difference in the proportion of collagen fiber contents in mouse livers among the four groups (F = 26.060, P < 0.001), and a higher proportion of collagen fiber contents was detected in mouse livers in the 4⁃week infection group [(11.29 ± 2.58)%] than in the control group (P < 0.001). Immunohistochemical staining revealed activation of a few HSCs and ECM deposition in mouse livers 1 and 2 weeks post⁃infection, and abundant brown⁃yellow stained α⁃SMA and COL1A1 were deposited in the lesion areas in mouse livers 4 weeks post⁃infection, which spread to surrounding tissues. Semi⁃quantitative analysis revealed significant differences in α⁃SMA (F = 7.667, P < 0.05) and COL1A1 expression (F = 6.530, P < 0.05) in mouse levers among the four groups, with higher α⁃SMA [(7.13 ± 3.68)%] and COL1A1 expression [(13.18 ± 7.20)%] quantified in mouse livers in the 4⁃week infection group than in the control group (both P values < 0.05). Scanning electron microscopy revealed significant differences in the fenestration frequency (F = 37.730, P < 0.001) and porosity (F = 16.010, P < 0.001) on the surface of mouse LSECs among the four groups, and reduced fenestration frequency and porosity were observed in the 1⁃ [(1.22 ± 0.48) /μm2 and [(3.05 ± 0.91)%] and 2⁃week infection groups [(3.47 ± 0.10)/μm2 and (7.57 ± 0.23)%] groups than in the control group (all P values < 0.001). There was a significant difference in the average fenestration diameter on the surface of mouse LSECs among the four groups (F = 15.330, P < 0.001), and larger average fenestration diameters were measured in the 1⁃ [(180.80 ± 16.42) nm] and 2⁃week infection groups [(161.70 ± 3.85) nm] than in the control group (both P values < 0.05). In addition, there were significant differences among the four groups in terms of Stabilin⁃1 (F = 153.100, P < 0.001), Stabilin⁃2 (F = 57.010, P < 0.001), Ehd3 (F = 31.700, P < 0.001), CD209b (F = 177.400, P < 0.001), GATA4 (F = 17.740, P < 0.001), and Maf mRNA expression (F = 72.710, P < 0.001), and reduced mRNA expression of Stabilin⁃1, Stabilin⁃2, Ehd3, CD209b, GATA4 and Maf genes was quantified in three infection groups than in the control group (all P values < 0.001). Conclusions　E. multilocularis infections may induce capillarization of LSECs in mice, and result in a reduction in the expression of functional and phenotypic marker genes of LSECs, and capillarization of LSECs occurs earlier than activation of HSC and development of hepatic fibrosis.

Key words: Echinococcus multilocularis, Alveolar echinococcosis, Liver sinusoidal endothelial cell, Capillarization, Hepatic fibrosis

摘要： 目的　观察泡型棘球蚴病发展过程中肝窦内皮细胞（liver sinusoidal endothelial cells, LSEC）毛细血管化现象及与肝纤维化的关系，为探索LSEC在泡型棘球蚴病所致肝损伤和肝纤维化发生和转归中的作用机制奠定基础。方法　取40只C57BL/6小鼠（6～8周龄），随机分为对照组和感染1、2、4周组，每组各10只。感染1、2、4周组每只小鼠经肝脏直视穿刺注射接种2 000个多房棘球蚴原头节，对照组用相同方法注射等量磷酸盐缓冲液。在感染后1、2、4周，取小鼠肝脏，经苏木精⁃伊红（hematoxylin⁃eosin, HE）染色后观察肝脏病理变化，经Masson三色染色后对染色阳性区域面积进行半定量分析以判断肝纤维化程度，采用免疫组织化学染色法评价α⁃平滑肌动蛋白（α⁃smooth muscle actin, α⁃SMA）和Ⅰ型胶原蛋白α1（collagen type I alpha 1, COL1A1）表达水平以判断肝星状细胞（hepatic stellate cell, HSC）活化和细胞外基质（extracellular matrix, ECM）沉积，用扫描电子显微镜观察LSEC表面窗孔。从小鼠肝脏中分离原代LSEC，用实时荧光定量PCR（real⁃time fluorescence quantitative PCR, qPCR）法检测LSEC标志基因稳定素（Stabilin）⁃1、Stabilin⁃2、Ehd3、CD209b、 GATA4、Maf mRNA表达水平。结果　多房棘球蚴感染2周后，可见小鼠肝脏局部肝小叶结构被破坏；感染4周后，小鼠肝脏可见多房棘球蚴囊泡，周围有肉芽肿组织包绕。Masson三色染色半定量分析结果显示，4组小鼠肝脏内胶原纤维比例差异有统计学意义（F = 26.060，P < 0.001），感染4周组小鼠肝脏内胶原纤维比例 [（11.29 ± 2.58）%] 较对照组增加（P < 0.001）。免疫组织化学染色结果显示，感染1、2周组小鼠肝脏内见少量HSC活化和ECM沉积；感染4周组小鼠肝组织中可见大量棕黄色α⁃SMA 和COL1A1聚集沉积在病变区域，并向周围扩散。半定量分析结果显示，4组小鼠肝脏内α⁃SMA和COL1A1表达水平差异均有统计学意义（F = 7.667、6.530，P 均 < 0.05），感染4周组小鼠肝脏内α⁃SMA [（7.13 ± 3.68）%]和COL1A1[（13.18 ± 7.20）%] 表达水平均较对照组增加（P 均< 0.05）。扫描电子显微镜结果显示，4组小鼠LSEC开窗频率和孔隙率差异均有统计学意义（F = 37.730、16.010，P 均 < 0.001），感染1 [分别为（1.22 ± 0.48）/μm2、（3.05 ± 0.91）%]、2周组 [分别为（3.47 ± 0.10）/μm2、（7.57 ± 0.23）%] 小鼠LSEC开窗频率和孔隙率均较对照组降低（P 均< 0.001）。4组小鼠LSEC平均窗孔直径差异有统计学意义（F = 15.330，P < 0.001），感染1 [（180.80 ± 16.42） nm]、2周组 [（161.70 ± 3.85） nm] 小鼠LSEC平均窗孔直径均较对照组增加（P 均 < 0.05）。4组小鼠LSEC中Stabilin⁃1、Stabilin⁃2、Ehd3、CD209b、GATA4、Maf mRNA表达水平差异均有统计学意义（F = 153.100、57.010、31.700、177.400、17.740、72.710，P均< 0.001）；与对照组相比，感染1、2、4周组小鼠上述基因相对表达量均降低（P均 < 0.001）。结论　多房棘球蚴感染可诱导小鼠LSEC发生毛细血管化，并使LSEC功能和表型标志基因表达水平降低；LSEC毛细血管化早于HSC活化及肝纤维化发生。 　

关键词: 多房棘球蚴, 泡型棘球蚴病, 肝窦内皮细胞, 毛细血管化, 肝纤维化