摘要
目的 观察ICOS转基因小鼠感染日本血吸虫后原代肝星状细胞(HSCs)的活化效应,探讨ICOSL/ICOS信号介导HSCs活化在血吸虫病肝纤维化形成中的作用.方法 建立ICOS-Tg小鼠及野生型小鼠日本血吸虫模型,通过肝脏酶灌注法和密度梯度离心法分离感染前(0周)和感染后(4~9周)的小鼠原代HSCs,运用台盼蓝拒染法鉴定分离的原代HSCs成活率;应用其在328 nm波长紫外激发光下自发荧光特性以及在肝细胞中其特异性表达神经胶质原纤维酸性蛋白(GFAP)鉴定分离的原代HSCs纯度;培养7d后,采用SYBR染料法实时荧光定量PCR检测原代HSCs中TGF-β1,Ⅰ、Ⅲ型胶原及α-SMA mRNA的表达变化.结果 分离的原代HSCs纯度达到90%,其成活率为95%.ICOS-Tg小鼠肝HSCs中α-SMAmRNA表达水平即HSCs的活化程度在感染后6、9周显著高于同时期野生型小鼠(P<0.01).ICOS-Tg小鼠肝HSCs中TGF-β1,Ⅰ、Ⅲ型胶原mRNA水平亦高于同时期野生型小鼠的水平,特别是在感染后6、9周呈显差异有统计学意义(P<0.01~0.05).结论 与野生型小鼠相比,感染日本血吸虫的ICOS-Tg小鼠肝HSCs活化增强,显著上调TGF-β1 mRNA、Ⅰ、Ⅲ型胶原mRNA及α-SMA mRNA的表达,提示在ICOS-Tg小鼠,ICOSL/ICOS信号的增强可明显上调HSCs活化促进肝纤维化的形成.
We observed the impact of the expression of TGF-β1 and collagen Ⅰ,Ⅲ in hepatic stellate cells in ICOS transgenic mice infected with Schistosoma japonicum,and explored the new effective way for controlling the development of hepatic fibrosis.ICOS transgenic (ICOS-Tg) mice and wild-type mice as experimental schistosomiasis model for Schistosoma japonicum were established.The liver ot ICOS-Tg mice was perfused in situ successively under agitation.HSCs were then separated by density gradient centrifugation through 16% and 12% OptiPrep on day before infection 0 week,and at 4,6 and 9 weeks post-infection.Cells viability was tested by erythrosin exclusion.The purity of HSCs was assessed by fluorescence of retinoid-containing vacuoles under ultraviolet excitation and immunocytochemisy of GFAP,which was a specific marker of HSCs not expressed in other liver cell types.Total RNA of primary HSCs cultured for 7 days was extracted by using TRIzol reagent.Real-time quantitative PCR was performed using SYBR Premix Ex Taq to assess the expression levels in primary HSCs Collagen-Ⅰ,Collagen-Ⅲ,α-SMA,TGF-β1,and other genes in the development of schistosomiasis.Purity of HSCs isolates was higher than 90%,and cell viability exceeded 95%.Cells were in good condition during the primary culture.With the develop of schistosomiasis,the degree of activation of HSCs was increasing,and the expression of Collagen-Ⅰ,Collagen-Ⅲ,α-SMA,and TGF-β1 in HSCs were enhancing.The level of HSCs activation in ICOS-Tg mice was significantly higher than that in wild-type mice (P<0.01).Also,the expression on Collagen-Ⅰ,Collagen-Ⅲ,α-SMA,and TGF-β1 in HSCs of ICOS-Tg mice were higher than that in wild-type mice (P<0.01-0.05).The co-stimulatory molecules ICOSL/ICOS signaling pathway has an important impact on the process of HSCs activation and fibrosis formation in mice infected with Schistosoma japonicum.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2014年第11期1103-1109,共7页
Chinese Journal of Zoonoses
基金
国家自然科学基金项目(No.81171603)资助~~