1、 中国现代应用药学 2023 年 1 月第 40 卷第 2 期 Chin J Mod Appl Pharm,2023 January,Vol.40 No.2 163 基于生信学数据挖掘和实验验证阿魏酸介导间充质干细胞外泌体调控炎症 miRNA 表达情况 熊武1，肖郁婷2,3a，杨莹3a，彭阿建3a，赵世情3b，谭梅鑫3a，段文丽1，肖慧1，梁文菲3a，张熙2*(1.湖南中医药大学第一附属医院，长沙 410007；2.湖南省脑科医院，长沙 410007；3.湖南中医药大学，a.中西医结合学院，b.针灸推拿与康复学院，长沙 410208)摘要：目的 基于生信分析筛选出人脐血间充质干细胞来源的外泌体
2、(human umbilical cord blood mesenchymal stem cells-derived exosomes，hUCBMSC-Exos)中可用于调控炎症过程的微小 RNA(microRNA，miRNA)。在此基础上，研究阿魏酸(ferulic acid，FA)对 hUCBMSC-Exos 中与炎症调控相关 miRNA 的影响。方法 从现有数据库中分别筛选hUCBMSC-Exos 中的 miRNAs 和与调控炎症相关的 miRNAs，取 2 组 miRNAs 的交集，从而获得 hUCBMSC-Exos 中与调控炎症相关的候选 miRNA 分子组。取足月健康新生儿脐带血，
3、将分离所得的单个核细胞置于 DMEM/F12 培养基培养至P4 代细胞，进行成骨、成软骨和成脂诱导分化鉴定。实验分为 2 个部分来验证 FA 对 hUCBMSC-Exos 及 hUCBMSCs 分泌相关炎症因子的作用：将鉴定成功的 hUCBMSC-Exos 随机分为实验组及对照组，实验组用 2 mgL1 FA 干预，对照组用等体积 PBS 液处理，培养 24 h。采用超速离心法结合超滤法提取 hUCBMSC-Exos，通过透射电子显微镜观察其形态，蛋白印迹法鉴定其特征表面标志物 CD9、CD63 和 TSG101，采用 BCA 法测定 hUCBMSC-Exos 浓度，RT-qPCR 法检测各组
4、外泌体中炎症调控相关候选 miRNAs 的表达。另将鉴定成功的人 hUCBMSCs 按随机数字表法随机分为实验组、模型组和对照组，实验组与模型组用含 30 mmo1L1的葡萄糖 DMEM/F12 培养基培养 120 h，建立高糖诱导损伤 hUCBMSCs细胞模型后，实验组用 2 mgL1 FA 干预，模型组和对照组用等容量的 PBS 液处理。每组均培养 24 h 后用 ELISA 试剂盒检测 3 组上清液中炎性因子含量。结果 通过对 GEO 测序数据分析和对文献的筛选最终获得 7 个在 hUCBMSC-Exos 中可能高表达且与炎症调控相关的 miRNAs：miR-125b、miR-126、m
5、iR-145、miR-146a、miR-21、miR-221、miR-31-5p。成功培养、分离和鉴定得到 hUCBMSCs，并进一步收集到 hUCBMSC-Exos，培养 24 h 后，实验组 hUCBMSC-Exos 的浓度为(1.1790.03)gmL1，明显高于对照组(0.8810.03)gmL1(P0.01)。与对照组相比，实验组中部分炎症调控相关miRNA 变化显著，miR-126-3p 和 miR-146a-5p 显著上升，miR-145 及 miR-31-5p 显著下降，差异有统计学意义(P0.05)。与模型组相比，在 2 mgL1 FA 干预下，实验组中 hUCBMSCs 分
6、泌 IL-1、MCP-1 水平明显降低，分泌 IL-10、M-CSF明显水平升高，差异具有统计学意义(P0.05)，2 组间 IL-6 分泌差异无统计学意义。与对照组相比，模型组 hUCBMSCs分泌 IL-1、IL-6、MCP-1 水平明显增高，差异具有统计学意义(P0.05)，2 组间 IL-10、M-CSF 分泌差异无统计学意义。结论 FA 可能通过介导 hUCBMSC-Exos 中调控炎症的 miRNA(miR-126-3p、miR-146a-5p、miR-145 及 miR-31-5p)分泌，进一步调控 hUCBMSCs 分泌相关炎症因子从而调控机体炎症反应，本研究进一步从外泌体水平
7、证实 FA 具有调控炎症的潜能。为进一步研究 FA 通过 hUCBMSC-Exos 调控机体炎症反应奠定坚实基础。关键词：阿魏酸；间充质干细胞；外泌体；炎症；miRNA；医学信息学 中图分类号：R285.4 文献标志码：A 文章编号：1007-7693(2023)02-0163-09 DOI:10.13748/ki.issn1007-7693.2023.02.003 引用本文：熊武,肖郁婷,杨莹,等.基于生信学数据挖掘和实验验证阿魏酸介导间充质干细胞外泌体调控炎症 miRNA表达情况J.中国现代应用药学,2023,40(2):163-171.Verification of Inflammato
8、ry miRNA Expression Regulated by Ferulic Acid-mediated hUCBMSC-Exos Based on Bioinformatics Data Mining and Experiment XIONG Wu1,XIAO Yuting2,3a,YANG Ying3a,PENG Ajian3a,ZHAO Shiqing3b,TAN Meixin3a,DUAN Wenli1,XIAO Hui1,LIANG Wenfei3a,ZHANG Xi2*(1.The First Hospital of Hunan University of Chinese Me
9、dicine,Changsha 410007,China;2.Hunan Brain Hospital,Changsha 410007,China;3.Hunan University of Chinese Medicine,a.College of Integrated Traditional Chinese and Western Medicine,b.College of Acupuncture&Tuina and Rehabilitation,Changsha 410208,China)基金项目：国家自然科学基金青年基金项目(81904217)；湖南省科技厅临床医疗技术创新引导项目(2
10、021SK50802)；湖南省卫生健康委科研计划项目(20201388，202203064523)作者简介：熊武，男，硕士，主治医师 E-mail: *通信作者：张熙，女，博士，教授 E-mail: 164 Chin J Mod Appl Pharm,2023 January,Vol.40 No.2 中国现代应用药学 2023 年 1 月第 40 卷第 2 期 ABSTRACT:OBJECTIVE To screen microRNAs(miRNAs)from human umbilical cord blood mesenchymal stem cells-derived exosomes(
11、hUCBMSC-Exos)that can be used to regulate inflammatory processes,and study the effect of ferulic acid(FA)on miRNAs related to inflammation regulation in hUCBMSC-Exos on this basis.METHODS miRNAs in hUCBMSC-Exos and miRNAs related to inflammation regulation were screened from the existing database,an
12、d their intersection was taken to obtain candidate miRNA molecular groups related to inflammation regulation in hUCBMSC-Exos.The umbilical cord blood of full-term healthy newborns was taken,and the isolated mononuclear cells were cultured in DMEM/F12 medium to P4 generation cells,and the differentia
13、tion of osteogenesis,chondrogenesis and adipogenesis was identified.The experiment included two parts to verify the effect of FA on hUCBMSC-Exos and inflammatory cytokines secreted by hUCBMSCs:The identified hUCBMSC-Exos were randomly divided into experimental group and control group.The experimenta
14、l group was intervened with 2 mgL1 FA,and the control group was treated with equal volume PBS solution for 24 h.hUCBMSC-Exos was extracted by ultracentrifugation combined with ultrafiltration,and its morphology was observed by transmission electron microscope.Its characteristic surface markers CD9,C
15、D63 and TSG101 were identified by Western blotting.The concentration of hUCBMSC-Exos was determined by BCA method,and the expression of candidate miRNAs related to inflammation in exosomes of each group was detected by RT-qPCR.The hUCBMSCs were randomly divided into experimental group,model group an
16、d control group according to the random number table method.The experimental group and model group were cultured in DMEM/F12 medium containing 30 mmo1L1 glucose for 120 h to establish the high glucose damaged hUCBMSCs model.The experimental group was treated with 2 mgL1 FA.Model group and control group were treated with PBS solution of equal volume.The content of inflammatory factors in supernatant of each group was detected by ELISA kit after 24 h culture.RESULTS The 7 miRNAs such as miR-125b,m
