脐带间充质干细胞改善自身免疫性肝炎免疫微环境的机制  

Mechanisms for improving the immune microenvironment of autoimmune hepatitis by umbilical cord mesenchymal stem cells

作　　者：余丽娜 阎升光 谢丹 高升[3] 叶政 陈姿任 王梓桦 欧阳石 YU Li′na;YAN Shengguang;XIE Dan;GAO Sheng;YE Zheng;CHEN Ziren;WANG Zihua;OUYANG Shi(School of Public Health,North China University of Science and Technology,Tangshan 063210,China;不详)

机构地区：[1]华北理工大学公共卫生学院,河北唐山063210 [2]广东高校生物靶向诊治与康复重点实验室,广州医科大学附属第五医院感染科,广州510700 [3]广东高校生物靶向诊治与康复重点实验室,广州医科大学附属第五医院中医科,广州510700 [4]广东高校生物靶向诊治与康复重点实验室,广州医科大学附属第五医院超声科,广州510700 [5]广东高校生物靶向诊治与康复重点实验室,广州医科大学附属第五医院急诊科,广州510700

出　　处：《实用医学杂志》2023年第12期1466-1472,共7页The Journal of Practical Medicine

基　　金：国家自然科学基金资助项目(编号:81803884);广州医科大学2022年学生创新能力提升计划项目(编号:02⁃408⁃2304⁃19064XM);2022年广州医科大学学科建设项目(编号:02⁃410⁃2206013);广州医科大学本科生能力提升创新项目(编号:2022JXA003);2023年市校(院)企联合资助项目(编号:2023A03J0421);广州市医学重点学科【2021⁃2023】项目;广东高校生物靶向诊治与康复重点实验室(编号:2021KSYS009)。

摘　　要：目的探讨脐带间充质干细胞(umbilical cord mesenchymal stem cells,UCMSCs)对自身免疫性肝炎(autoimmune hepatitis,AIH)免疫微环境的影响及潜在的调控机制。方法将AIH患者外周血单个核细胞(PBMCs)与UCMSCs分别以10∶1(Co⁃culture 1)或5∶1(Co⁃culture 2)的比例共培养。流式细胞术检测Th17、Treg及Th17/Treg比值;ELISA检测上清中转化生长因子β1(TGF⁃β1)、白细胞介素⁃17(IL⁃17)和IL⁃6的水平。运用RNA⁃seq筛选PBMCs共培养前后的差异基因,富集前15个表达量最高的mRNA进行后续生物信息分析。Western blot和RT⁃qPCR测定Foxp3、RORγt表达水平。结果UCMSCs显著降低CD3^(+)CD8^(-)IL⁃17A^(+)Th17比例,增加CD25^(+)Foxp3^(+)CD4^(+)Treg比例。与PBMCs组相比,Co⁃culture 2组的TGF⁃β1显著上调(P<0.01),IL⁃17和IL⁃6显著下调(P<0.05)。转录组测序分析发现转录因子主要集中在免疫应答及T细胞激活等领域;其中SMAD3和STAT3占据较核心的地位。蛋白和mRNA检测显示,Co⁃culture 2组Foxp3和SMAD3的表达水平显著上调(P<0.05),RORγt和STAT3的表达水平显著下调(P<0.05)。结论UCMSCs通过TGF⁃β通路促进Th17向Treg细胞分化,抑制炎症反应改善肝脏免疫微环境。Objective To investigate the impact and potential regulatory mechanisms of Umbilical Cord Mesenchymal Stem Cells(UCMSCs)on the immune microenvironment of autoimmune hepatitis(AIH).Methods Peripheral blood mononuclear cells(PBMCs)from AIH patients co⁃cultured with UCMSCs at a ratio of 10∶1(Co⁃culture 1)or 5∶1(Co⁃culture 2),respectively.Flow cytometry was used to detect Th17,Treg and Th17/Treg ratio;ELISA was used to detect the levels of transforming growth factorβ1(TGF⁃β1),interleukin⁃17(IL⁃17)and IL⁃6 in the supernatant.RNA⁃seq was used to screen the differential genes before and after co⁃culture of PBMCs,and the top 15 most highly expressed mRNAs were enriched for subsequent bioinformatic analysis.western blot and RT⁃qPCR were used to determine the expression levels of Foxp3 and RORγt.Results UCMSCs significantly decreased CD3^(+)CD8^(⁃)IL⁃17A^(+)Th17 ratio and increased CD25^(+)Foxp3^(+)CD4^(+)Treg ratio.Compared with the PBMCs group,TGF⁃β1 was significantly upregulated(P<0.01)and IL⁃17 and IL⁃6 were significantly downregu⁃lated(P<0.05)in the Co⁃culture 2 group.Transcriptome sequencing analysis revealed that the transcription factors were mainly focused on immune response and T cell activation;among them,SMAD3 and STAT3 occupied a more central position.Protein and mRNA assays showed that the expression levels of Foxp3 and SMAD3 were significantly upregulated in the Co⁃culture 2 group(P<0.01),and the expression levels of RORγt and STAT3 were significantly downregulated(P<0.01).Conclusion UCMSCs promote the differentiation of Th17 to Treg cells through the TGF⁃βpathway,suppress inflammatory responses to improve the hepatic immune microenvironment.

关 键 词：UCMSCs/PBMCs共培养系统 肝脏免疫微环境 RNA⁃seq Th17/Treg平衡 TGF⁃β信号通路 

分 类 号：R575.1[医药卫生—消化系统]