摘要
目的:探究背根神经节持续受压致神经病理性疼痛大鼠血浆外泌体中差异表达的miRNAs及其功能。方法:采用超速离心法提取并纯化健康对照大鼠及背根神经节持续受压大鼠血浆外泌体,通过透射电镜获取外泌体形态,采用Western Blot法进行外泌体标记蛋白鉴定;提取外泌体总RNA,Illumina HiSeq高通量测序检测miRNAs表达谱;生物信息学分析背根神经节持续受压后外泌体miRNAs的差异表达,预测其靶基因并分析其靶基因的潜在功能。结果:透射电镜、纳米粒径分析及Western Blot的结果均显示外泌体提取成功,电镜下形态为典型茶托样,平均粒径为136.2±1.56nm,标记蛋白TSG101、CD9均为阳性;高通量测序分析显示,实验组有152个血浆外泌体来源miRNAs表达显著改变,其中104个表达显著上调,48个表达显著下调。GO分析显示靶基因功能富集细胞器、细胞质上,与离子结合、ATP结合、催化活性等功能相关,参与系统的发展、细胞器物质代谢过程等。差异表达miRNA靶基因的KEGG通路分类统计显示在细胞间紧密连接、RAP1信号通路、PI3K-AKt信号通路及轴突导向、突触囊泡周期等存在靶基因的功能富集。结论:血浆外泌体miRNAs在背根神经节持续受压大鼠中的表达谱较健康对照组相比具有显著差异,推测差异表达的miRNAs靶基因在代谢、细胞间联系及神经可塑性等方面发挥作用。
Objective:To investigate the differentially expressed miRNAs and their functions in plasma exosomes of rats with neuropathic pain caused by continuous compression of dorsal root ganglion.Method:The plasma exosomes of healthy control rats and rats with chronic compression of dorsal root ganglion were purified by ultracentrifugation and identified by transmission electron microscope and Western Blot;The total RNA of exosomes was extracted,and the miRNAs expression profile was detected by Illumina HiSeq high-throughput sequencing;Bioinformatics was used to analyze the differentially expressed miRNAs between the two groups and predict their target genes and potential functions.Result:The exosome images were obtained by transmission electron microscope.The peak value of exosome particle size was 136.2±1.56nm by nanoparticle size detector.The exosome tag proteins TSG101 and CD9 were positive by Western Blot;High throughput sequencing analysis showed that the expression of 152 plasma secrete derived miRNAs in test group changed significantly,of which 104 were significantly up-regulated and 48 were significantly down regulated.Go analysis shows that the target gene functions are enriched in organelles and cytoplasm,related to ion binding,ATP binding,catalytic activity and other functions,and participate in the development of the system and the metabolic process of organelles.The classified statistics of KEGG pathway differentially expressing miRNA target genes showed that there was functional enrichment of target genes in intercellular tight junction,Rap1 signal pathway,PI3K Akt signal pathway,axon guidance and synaptic vesicle cycle.Conclusion:The expression profile of miRNAs in plasma exosomes of rats with chronic compression of dorsal root ganglion is significantly different from that of healthy controls.It is speculated that the differentially expressed miRNAs target genes play a role in metabolism,intercellular communication and neural plasticity.
作者
曲玉娟
续晓倩
岳寿伟
QV Yujuan;XU Xiaoqian;YUE Shouwei(Qilu Hospital of Shandong University,Jinan,250012)
出处
《中国康复医学杂志》
CAS
CSCD
北大核心
2023年第4期433-440,共8页
Chinese Journal of Rehabilitation Medicine
基金
国家自然科学基金项目(81702224)。
