日本血吸虫胞外囊泡的提取、鉴定及对人肝星状细胞影响的初探  

Extraction and Identification of Schistosoma japonicum Extracellular Vesicles and Its Effect on Human Hepatic Stellate Cells

作　　者：钟昊然 朱丹霖 董博文 陆珂[1] 李浩[1] 傅志强[1] 刘金明[1] 金亚美[1] ZHONG Haoran;ZHU Danlin;DONG Bowen;LU Ke;LI Hao;FU Zhiqiang;LIU Jinming;JIN Yamei(National Reference Laboratory for Animal Schistosomiasis,Shanghai Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Shanghai 200241,China)

机构地区：[1]中国农业科学院上海兽医研究所,国家动物血吸虫病参考实验室,上海200241

出　　处：《复旦学报（自然科学版）》2024年第3期398-404,414,共8页Journal of Fudan University：Natural Science

基　　金：上海市自然科学基金(20ZR1469300);国家自然科学基金(31672245);中国农业科学院科技创新工程(CAAS-ASTIP-2021-SHVRI)。

摘　　要：旨在探讨日本血吸虫(Schistosoma japonicum)胞外囊泡(Extracellular vesicles,EVs)的提取方法及特性。本研究利用尺寸排阻法收集Sj EVs,通过透射电镜、蛋白质谱对Sj EVs的形态学、蛋白谱进行解析并对鉴定到的蛋白进行生物信息学分析。将PKH67绿色荧光标记的Sj EVs与人肝星状细胞LX-2共孵育,观察细胞摄取Sj EVs的情况,并对细胞中虫源miRNAs及细胞内纤维化因子进行检测,初步探讨Sj EVs对肝星状细胞的影响。结果表明:Sj EVs呈圆形或椭圆形囊泡结构,内含EVs标志蛋白。生物信息学分析结果显示,Sj EVs蛋白可能与细胞进程、代谢等途径密切相关。Sj EVs与人肝星状细胞LX-2共孵育结果显示,LX-2细胞可成功摄取Sj EVs,且定量反转录PCR(RT-qPCR)结果显示LX-2细胞中部分虫源miRNAs显著升高,纤维化因子α-平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原纤维α1(Collagen typeⅠalpha 1,Col1α1)和Ⅲ型胶原纤维α1(Collagen typeⅢalpha 1,Col3α1)极显著升高,提示肝星状细胞活化。综上,本研究为解析Sj EVs在宿主肝纤维化中的作用提供了依据,为进一步探究Sj EVs的潜在功能奠定了基础。To explore the isolation technique and characterization of extracellular vesicles(EVs)of Schistosoma japonicum,in the present study,the size exclusion method was utilized to collect Sj EVs,and the morphology and protein profiles of Sj EVs were analyzed by transmission electron microscopy and protein mass spectrometry,respectively.Bioinformatics analysis was conducted on all identified proteins.PKH67 green fluorescence labeled Sj EVs were co-incubated with human hepatic stellate cell line(LX-2)to observe the uptake of Sj EVs and the expression of Sj-miRNAs and the fibrotic factors in the cells were also detected.The results showed that Sj EVs presented vesicular structures and contained EVs marker proteins.Bioinformatics analysis showed that Sj EVs proteins might be closely related to cellular processes and metabolism pathways.Co-incubation of Sj EVs with LX-2 cells revealed that Sj EVs could be successfully absorbed,and significantly increased expression of Sj-miRNAs and fibrotic factors were also observed.In summary,this study provides a theoretical basis for analyzing the potential functions of Sj EVs,with a view to laying a foundation for further elucidation of schistosome-host interactions.

关 键 词：日本血吸虫 胞外囊泡 MICRORNA 人肝星状细胞 

分 类 号：S858[农业科学—临床兽医学]