经门静脉注射虫卵诱导血吸虫肝病小鼠模型的构建与评价

Establishment and evaluation of a mouse model of schistosomiasis liver disease induced by portal vein injection of worm eggs

目的构建经门静脉直接注射日本血吸虫虫卵诱导血吸虫肝病小鼠模型并评价其效果,为血吸虫肝病研究提供新的动物模型。方法将15只8周龄的C57BL/6雄性小鼠随机分为对照组和注射虫卵组,其中对照组5只,注射虫卵组10只。第-14天将5000个活虫卵注射至小鼠腹腔,第0天麻醉小鼠后剖开腹腔,经门静脉注入5000个虫卵,对照组注射等体积的磷酸盐缓冲盐溶液(PBS)。分别于第10天、第30天处死虫卵组小鼠各5只,第10天处死对照组小鼠,收集小鼠血清和肝组织,通过进行苏木素-伊红染色(HE染色)、马松染色(Masson染色),微孔板分光光度计法检测丙氨酸转氨酶(alanine aminotransferase,ALT)、天门氨酸转氨酶(aspartate aminotransferase,AST)、肝脏羟脯氨酸(hydroxyproline,HYP)含量,以及实时荧光定量PCR(qPCR)法检测肝纤维化相关基因、Th1和Th2型免疫反应相关基因,分析肝损伤、虫卵肉芽肿和纤维化以及适应性免疫反应等指标,评价经门静脉注射虫卵诱导血吸虫肝病小鼠模型的效果。结果经门静脉注射虫卵10 d和30 d后,小鼠肝脏出现明显的虫卵肉芽肿,第10天组虫卵肉芽肿面积与第30天组相比,差异无统计学意义(t=0.975,P=0.332)。Masson染色和肝脏羟脯氨酸含量检测显示肝脏发生明显纤维化。qPCR结果表明,与对照组相比,第10天组小鼠肝组织纤维化标志基因α-平滑肌肌动蛋白(alpha smooth muscle actin,α⁃Sma)、Ⅰ型胶原(collagen type I alpha 1,Col1α1)、转化生长因子β1(transforming growth factor beta 1,Tgfb1)表达水平显著升高,差异有统计学意义(t=6.380、7.533、5.314,P=0.002、0.001、0.007);在第30天时出明显下降,与对照组相比差异无统计学意义(t=0.940、1.529、1.746,P=0.778、0.543、0.457)。同时,Th1型免疫反应相关基因肿瘤坏死因子α(tumor necrosis factor alpha,Tnf)、干扰素-γ(interferon gamma,Ifng)和Th2型免疫反应相关基因白细胞介素5(interleu⁃kin-5,Il5)、白细胞介素13(interleukin-13,Il13)表达水平在注射虫卵10 d后显著升高,差异有统计学意义(t=6.163、4.589、5.651、5.367,P=0.003、0.018、0.020、0.009)。此外,各组小鼠血清中的AST、ALT水平无明显变化,差异无统计学意义(t=0.982、3.450,P=0.771、0.074;t=1.164、0.564,P=0.697、0.917)。结论经门静脉注射虫卵诱导血吸虫肝病小鼠模型构建成,研究结果为血吸虫病肝纤维化的机制研究提供了新的建模方法。

Objective To construct a mouse model of Schistosoma japonicum liver disease induced by direct injection of Schistosoma japonicum eggs through the portal vein and evaluate its effectiveness,in order to provide a new animal model for schistosomiasis liver disease research.Methods Fifteen 8-week-old C57BL/6 male mice were randomly divided into control group and egg injection group,with 5 in the control group and 10 in the egg injection group.On day-14,5000 live eggs were injected into the abdominal cavity of mice,and on day 0,the mice were anesthetized and the abdominal cavity was opened.5000 live eggs were injected through the portal vein,and the control group was injected with equal volume of phosphate buffer(PBS).5 mice in the egg group were killed on day 10 and 30,respectively.The control group mice were killed on day 10,and their serum and liver tissue were collected.Hematoxylin eosin staining(HE)and Masson staining were performed,and alanine aminotransferase(ALT),aspartate aminotransferase(AST),and liver hydroxyproline(HYP)content were detected using a microplate spectrophotometer.Liver fibrosis-related genes,Th1 and Th2 type immune response-related genes were analyzed by real-time fluorescence quantitative PCR(qPCR).Liver injury,egg granuloma and fibrosis,and adaptive immune response were detected to evaluate the effect of portal vein injection of eggs while inducing mouse model of schistosomiasis liver disease.Results The results showed that significant egg granulomas appeared in the liver of mice after injection of eggs into the portal vein for 10 and 30 days.There was no statistically significant difference in the area of egg granulomas between the 10-day group and the 30-day group(t=0.975,P=0.332).Masson staining and liver hydroxyproline content detection showed significant fibrosis in the liver.The qPCR results showed that,compared with the control group,the expression levels of fibrosis marker genes,such asα⁃Sma(alpha smooth muscle actin),Col1a1(collagen typeⅠalpha 1),and Tgfb1(transforming growth factor beta 1),were significantly increased(t=6.380,7.533,5.314;P=0.002,0.001,0.007),and then decreased on the 30th day,with no statistical difference compared to the control group(t=0.940,1.529,1.746;P=0.778,0.543,0.457).At the same time,the expression levels of Th1 type immune response-related genes,such as tumor necrosis factor alpha(Tnf),interferon gamma(Ifng),and Th2 type immune response-related genes,such as interleukin-5(Il5),interleukin-13(Il13),significantly increased 10 days after eggs injection(t=6.163,4.589,5.651,5.367;P=0.003,0.018,0.020,0.009).In addition,there was no significant change in the levels of AST and ALT in the serum of each group of mice(t=0.982,3.450;P=0.771,0.074.t=1.164,0.564;P=0.697,0.917).Conclusions A mouse model of schistosomiasis liver disease induced by portal vein injection of worm eggs was constructed.The study provides a new modeling method for studying the mechanism of liver fibrosis in schistosomiasis..