单性日本血吸虫成虫感染性小鼠肝纤维化研究

The study on mice hepatic fibrosis caused by adult unisexual Schistosoma japonicum infection

目的观察和评估单性日本血吸虫成虫感染小鼠出现肝纤维化病变风险。方法将40只小鼠随机分为双性感染组、单性雄虫组、单性雌虫组和未感染组,每组10只,分别经腹壁皮肤接触感染雌雄尾蚴、雄性尾蚴、雌性尾蚴和不含尾蚴的去氯水,感染尾蚴数为(40±1)条/只。双性感染组小鼠感染周期为6周,其余3组为36周。取小鼠肝脏,观察肝脏大体形态;经HE和Masson染色镜下观察小鼠肝脏病理变化和胶原纤维增生情况;测定肝脏胶原纤维特有的羟脯氨酸含量;应用荧光定量PCR和Western blot检测肝组织α-SMA、TGF-β1、Col1、Col3 mRNA和蛋白表达水平。结果与无感染组比较,单性雄虫组小鼠肝脏颜色发黑,肝组织内有大量色素沉积,周围有炎性细胞浸润,色素沉积处有胶原纤维;单性雌虫组小鼠肝脏大体形态与无感染组相似,病理变化和胶原纤维增生明显轻于单性雄虫组。单性雄虫组、单性雌虫组、双性感染组和未感染组小鼠的肝脏羟脯氨酸含量分别为(0.209±0.033)、(0.187±0.034)、(0.194±0.04)和(0.122±0.016)μg/mg,单性雄虫组、单性雌虫组均显著高于未感染组(P均<0.05),与双性感染组均无明显差异。单性雄虫组小鼠肝脏Col1(2.4±0.91)和α-SMA(0.22±0.1)mRNA表达都明显高于未感染组[Col1(1.05±0.29)、α-SMA(0.08±0.01)](P均<0.05),Col1(2.4±0.91)(P<0.01)和TGF-β1(4.28±2.58)(P<0.05)mRNA表达均显著高于双性感染组;单性雌虫组各基因mRNA表达水平与未感染组相比均无显著差异,Col1(1.34±0.18)和TGF-β1(3.55±1.34)mRNA表达水平也显著高于双性感染组[Col1(0.84±0.14)、TGF-β1(0.31±0.11)、α-SMA(0.36±0.11)、Col3(2.15±0.61)](P均<0.01),α-SMA(0.17±0.11)(P<0.05)和Col3(0.51±0.23)(P<0.01)mRNA表达则显著降低。单性雄虫组小鼠肝脏各基因蛋白表达水平与未感染组比较均无明显差异;单性雌虫组TGF-β1(1.03±0.39)蛋白表达量显著低于未感染组TGF-β1(1.78±0.29)(P<0.05)。结论单性血吸虫成虫会导致小鼠肝脏炎症细胞浸润,胶原纤维增生,羟脯氨酸增加、部分肝纤维化分子指标mRNA表达上调等肝纤维化特征和向肝纤维化演变趋势,并且单性雄虫比单性雌虫更易引发小鼠肝脏病变。

Objective To observe and evaluate the risks of liver fibrosis in mice infected with adult single-sex Schistosoma japonicum.Methods Forty mice were randomly divided into 4 groups:bisexual infection group,unisexual male group,unisexual female group and uninfected group,with 10 mice in each group.The 4 groups were respectively infected via abdominal skin contact with cercariae of both sexes,exclusively male cercariae,exclusively female cercariae and dechlorinated water without cercariae,with cercariae number being(40±1)per mouse.Mice in the bisexual infection group were sacrificed 6 weeks post-infection,whereas mice in the other 3 groups were sacrificed 36 weeks post-infection.The mice livers were sampled and observed for all the 4 groups;the pathological changes and collagen expression of liver specimens were detected using hematoxylin&eosin(HE)and Masson staining;the liver hydroxyproline content was determined;the mRNA and protein expression levels ofα-SMA,TGF-β1,Col1 and Col3 in liver tissues of each group were detected by fluorescence quantitative PCR and Western blot.Results Compared with the uninfected group,the liver color of the unisexual male group was darkened,there was marked pigment deposition in the liver tissue,surrounded by inflammatory cells infiltration and collagen fibers hyperplasia;the pathological changes and collagen fiber hyperplasia were much less prominent in the unisexual female group than in the unisexual male group.The liver hydroxyproline content of both unisexual male group(0.209±0.033)μg/mg and unisexual female group(0.187±0.034)μg/mg was significantly higher than that of the uninfected group(0.122±0.016)μg/mg(both P values<0.05),as high as that of the bisexual group(0.194±0.04)μg/mg.The mRNA expressions of Col1(2.4±0.91)andα-SMA(0.22±0.1)in the liver of the unisexual male group were significantly higher than those of the uninfected group(Col1(1.05±0.29)andα-SMA(0.08±0.01),both P values<0.05),and the mRNA expressions of liver Col1(2.4±0.91)(P<0.01)and TGF-β1(4.28±2.58)(P<0.05)of the unisexual male group were significantly higher than those of the bisexual infection group;There was no significant difference in mRNA expression levels of all 4 genes between the unisexual female group and the uninfected group,whereas the Col1(1.34±0.18)and TGF-β1(3.55±1.34)mRNA expression levels of the unisexual female group were significantly higher than those of bisexual infection group(Col1(0.84±0.14),TGF-β1(0.31±0.11),α-SMA(0.36±0.11)),Col3(2.15±0.61)(both P values<0.01),withα-SMA(0.17±0.11)(P<0.05)and Col3(0.51±0.23)(P<0.01)mRNA expressions significantly decreased.There was no significant difference in the protein expression levels of all 4 liver genes in the unisexual male group compared with the uninfected group,and the protein expression of TGF-β1(1.03±0.39)in unisexual female group was significantly lower than TGF-β1(1.78±0.29)expression in the uninfected group(P<0.05).Conclusion Adult single-sex Schistosoma japonicum causes liver pathological changes in mice including inflammatory cells infiltration,collagen fiber hyperplasia,increased hydroxyproline content,up-regulated mRNA expression level of genes related to hepatic fibrosis,indicating progression tendency to liver fibrosis,and the male schistosoma infection results in more severe damages to the mouse liver than the female schistosoma.