多房棘球蚴病患者肝病灶组织差异表达基因的研究

Investigation of differentially expressed genes in liver tissues of patients with alveolar echinococcosis

目的探讨多房棘球蚴感染小鼠肝差异基因在多房棘球蚴病患者肝病灶组织中的表达情况及意义。方法选取高通量基因表达数据库中编号为GSE24376的小鼠感染多房棘球蚴基因芯片数据集,分析感染小鼠和未感染小鼠之间的差异表达基因,对差异表达基因进行基因本体(GO)分析。取6例多房棘球蚴病患者肝病灶组织和距离病灶组织2 cm的周围肝组织,采用荧光定量PCR检测2个高表达差异基因mRNA相对转录水平。取45份多房棘球蚴病患者石蜡包埋肝组织样品(炎性反应和肝纤维化改变严重的病灶29份,较少炎性细胞浸润和肝纤维化病变的肝组织16份),采用免疫组化法分析2个高表达差异基因的表达情况。用SPSS 21.0统计学软件对数据进行统计分析。结果生物信息学分析结果显示,共筛选出21个差异表达基因,其中9个基因表达上调,12个基因表达下调。GO分析结果显示,差异基因主要位于细胞内囊泡及细胞外基质等细胞成分中,参与了中性粒细胞的脱颗粒及活化等生物过程,并且具有CC趋化因子受体结合活性及趋化因子活性。高表达差异基因CHI3L3 (在人体中为CHI3L1)和CCL8荧光定量PCR结果显示,6例多房棘球蚴病患者肝病灶组织中CHI3L1和CCL8 mRNA相对转录水平均高于病灶周围肝组织(P <0.01),最高倍数分别达7倍和9倍。免疫组化分析结果显示:29份多房棘球蚴病患者肝组织炎性反应和肝纤维化改变严重的病灶样品中,有20份(占69%)呈CHI3L1蛋白高表达,其主要着色部位位于细胞浆;有22份(占76%)呈CCL8蛋白高表达。16份多房棘球蚴病患者肝组织轻微炎症样品中,2份(占13%)呈CHI3L1蛋白阳性;4份(占33%)呈CCL8蛋白阳性。炎性反应和肝纤维化改变严重的病灶组织中,CHI3L1和CCL8蛋白的阳性表达率分别达96%和97%,均高于轻微炎症肝组织的3%和8%(P <0.01)。结论 CHI3L1和CCL8蛋白在多房棘球蚴病患者肝组织中表达增高,提示二者在机体对虫体的免疫应答过程中发挥了一定作用。

Objective To investigate the expression profile of differentially expressed genes in liver lesions of alveolar echinococcosis patients,based on the findings that these genes were indentified in mice infected with alveolar metacestode.Methods The gene chip dataset(No.GSE24376)in the high-throughput gene expression database was selected from the gene expression omnibus(GEO),to compare the genes with differential expression between infected and uninfected mice.The differentially expressed genes underwent gene ontology(GO)analysis.Lesion tissues and adjacent tissues 2 cm from the lesion were collected from 6 patients with E.multilocularis cyst infection.Fluorescence quantitative PCR was performed to detect the relative mRNA levels of two highly expressed differential genes.Forty-five paraffin-embedded liver tissue samples from patients with multilocular hydatid infection(29 samples with severe inflammatory response and liver fibrosis,and 16 with relatively normal liver tissue with less inflammatory cell infiltration and liver fibrosis)underwent immunohistochemistry to analyze the expression of the two highly expressed differential genes.Statistical analysis was performed on the data with SPSS 21.0 statistical software.Results As compared to the normal mice,21 differentially expressed genes were found,including 9 overexpressed genes and 12 downregulated genes in liver tissues of infected mice.The results of GO analysis showed that the differentially expressed genes were expressed in vesicle lumen,cytoplasmic vesicle lumen and extracellular matrix,participated in biological processes of neutrophil degranulation and activation,and possessed CC chemokine receptor binding activity and chemokine activity.The fluorescence quantitative PCR results showed that the mRNA expression levels of CHI3L3(CHI3L1 in humans)and CCL8,which had higher differential expression,were higher in the liver lesion tissues than in the surrounding tissues of the 6 patients with multilocular hydatid infection(P<0.01),reaching an utmost 7 and 9 folds,respectively.The results of immunohistochemistry showed that among the 29 lesion samples with severe inflammatory reaction and liver fibrosis,20 samples(69%)showed high expression of CHI3L1 protein,with staining mainly located in the cytoplasm,and 22 samples showed high levels of CCL8 protein(76%).Of the 16 relatively normal liver tissue samples,only 2(13%)were positive for CHI3L1 protein,and only 4 were positive for CCL8 protein(33%).In the lesions with severe inflammatory response and liver fibrosis,the positive expression rate of CHI3L1 and CCL8 proteins reached 96%and 97%,respectively,which were higher than 3%and 8%in normal liver tissues(P<0.01).Conclusion The expression of CHI3L1 and CCL8 was upregulated in the liver tissues of patients with E.multilocularis,suggesting that they play a role in the immune response to parasites.