日本血吸虫感染小鼠肝星状细胞差异表达microRNA的鉴定

Identification of deregulated microRNAs in hepatic stellate cells from mice infected with Schistosoma japonicum

目的筛选并验证在感染日本血吸虫小鼠肝星状细胞(hepatic stellate cells,HSCs)中差异表达的microRNA(miRNA)。方法 12只6周龄的BALB/c雄性小鼠随机分为正常对照组和感染日本血吸虫组,其中对照组3只,感染组9只。采用Ⅳ型胶原酶体外灌注、密度梯度离心和CD11b磁珠阴性分选相结合的方法,分离小鼠肝脏中的原代HSC。取正常小鼠和感染血吸虫后第42天、49天、56天小鼠的原代HSC miRNA,并利用定量PCR(quantitative PCR, qPCR)技术验证测序结果。结果通过miRNA测序,筛选出感染后HSC中差异表达的宿主miRNA共89条,其中75条表达上调,14条表达下调;qPCR验证部分表达改变miRNA的结果显示,miR-17-5p、miR-21a-5p、miR-25-3p、miR-100-5p、miR-122-5p的表达趋势与测序结果基本一致。结论日本血吸虫感染导致宿主HSC部分miRNA表达谱发生了明显改变,这些差异表达的miRNA在血吸虫病肝纤维化进展中起着重要调控作用,其可能在肝纤维化发生发展过程中参与对某些信号通路的调控,确切机制需进一步深入研究。本研究结果为这些miRNA在血吸虫病肝纤维化中的功能及其机制研究提供重要基础。

Objective To screen and validate differentially expressed microRNAs(miRNAs)in hepatic stellate cells(HSCs)from mice infected Schistosoma japonicum.Methods Twelve male BALB/c mice at 6 weeks of age were divided into the control group and infection group randomly,among which 3 were in the control group and 9 were in the infection group.The primary HSCs was isolated by collagenaseⅣdigestion,density gradient centrifugation and negatively sorting by CD11b magnetic microbeads.RNA sequencing was used to screen the aberrant miRNAs in primary HSCs from infected mice at day 42,49,56 post infection,then quantitative PCR(qPCR)was used to validate the result of RNA sequencing.Results RNA sequencing identified 89 differentially expressed miRNAs in HSCs during infection,of which 75 were up-regulated,and 14 were down-regulated.qPCR data showed that the expression levels of miR-17-5p,miR-21 a-5p,miR-25-3p,miR-100-5p and miR-122-5p were consistent with the sequencing results.Conclusions The expression profile of miRNAs in HSCs was obviously altered after infection.The differentially expressed miRNA has a vital role in the progress of schistosomal liver fibrosis.Many signal pathways of liver fibrosis may be regulated by miRNA and the exact mechanism needs further study.This result provides a basis for further investigation of their functions and mechanisms in the schistosomiasis hepatic fibrosis.