肿瘤坏死因子α改变人脐带间充质干细胞的生物学特性并降低其免疫调节能力  

Tumor necrosis factor-α alters the biological characteristics of human umbilical cord mesenchymal stem cell and reduces their immunomodulatory capacity

作　　者：胡彩东 林传明[1] 许希 谢水玲 李海亮[1] 刘礼平[1] HU Cai-dong;LIN Chuan-ming;XU Xi;XIE Shui-ling;LI Hai-liang;LIU Li-ping(Department of Hematology,the First Affiliated Hospital of Gannan Medical University,Ganzhou,Jiangxi 341000)

机构地区：[1]赣南医学院第一附属医院血液科,江西赣州341000

出　　处：《赣南医学院学报》2021年第9期881-886,共6页JOURNAL OF GANNAN MEDICAL UNIVERSITY

基　　金：江西省教育厅科学技术研究项目(GJJ180804);赣南医学院第一附属医院校级课题(YB201812)。

摘　　要：目的:研究肿瘤坏死因子α(Tumor necrosis factorα,TNF-α)对人脐带间充质干细胞表面抗原表达、成骨及成脂分化、增殖、免疫调节功能的影响。方法:采用酶消化、贴壁培养法从正常脐带组织中获取相对纯净人脐带间充质干细胞(Umbilical cord mesenchymal stem cells,hUC-MSC),实验组在培养基中加入TNF-α(10 ng·mL^(-1))刺激24 h,对照组培养基中无添加。(1)比较实验组与对照组hUC-MSC生长形态变化;(2)流式细胞术检测实验组与对照组hUC-MSCs表面分子表达;(3)分化培养基诱导实验组与对照组hUC-MSC成脂成骨分化,油红O染色、Von Kossa染色分别观测脂滴形成、钙盐沉积情况,鉴定其多向分化潜能;(4)MTS检测实验组与对照组hUC-MSCs增殖能力;(5)将PHA刺激的人外周血单个核细胞(Peripheral blood mononuclear cells,PBMC)与实验组及对照组hUC-MSCs共培养,ELISA检测上清中IFN-γ的分泌量;实时定量PCR检测刺激前后人脐带间充质干细胞吲哚胺2,3-双加氧酶(Indoleamine 2,3-dioxygenase,IDO)的表达。结果:(1)成功培养并传代高纯度人脐带间充质干细胞,实验组与对照组相比hUC-MSC生长形态未见明显改变;(2)与对照组相比,实验组hUC-MSCs表面CD54和CD106表达明显升高;(3)与对照组相比,实验组hUC-MSCs成脂分化能力未见明显差异,成骨分化实验钙盐沉积块较对照组小;(4)实验组hUC-MSCs增殖能力较对照组弱;(5)当hUC-MSCs∶hPBMC为1∶5时,TNF-α(10 ng·mL^(-1))刺激24 h后hUC-MSCs对活化的hPBMC的免疫抑制能力(即抑制IFN-γ的分泌)已表现出差异趋势,当比例扩大到1∶20时出现统计学差异,且刺激后人脐带间充质干细胞IDO表达下降。结论:TNF-α刺激可影响hUC-MSC表面分子表达、削弱其增殖能力,但不影响其多向分化功能;TNF-α可通过下调hUC-MSCs对IDO的表达而降低对hPBMC的免疫调节能力。Objective:To investigate the effects of tumor necrosis factorα(TNF-α)on human umbilical cord mesenchy⁃mal stem cells,including the expression of cell surface antigen,osteogenic and adipogenic differentiation,proliferation,and immunoregulatory capacity.Methods:HUC-MSCs digested from normal umbilical cord tissue were divided into 2 groups.One group as the experimental group was treated with TNF-α(10 ng·mL^(-1))for 24 hours,and the other one as the control group did not receive any treatment.(1)The morphological characteristics of hUC-MSCs were compared be⁃tween 2 groups;(2)The expression of surface molecular on hUC-MSCs of 2 groups was detected by flow cytometry;(3)hUC-MSCs of both groups were cultured in differentiation medium.Their osteogenic and adipogenic differentiation potential were identified by observing the formation of lipid droplets and the deposition of calcium salts using oil red staining and Von kossa staining;(4)MTS was used to detect the proliferation of hUC-MSCs in the two groups;(5)Human pe⁃ripheral blood mononuclear cells(PBMCs)stimulated by PHA were co-cultured with hUC-MSCs of experimental group and control group,respectively.IFN-γsecretion in the supernatant was detected by ELISA;IDO expression on hUCMSCs before and after stimulation was detected by real-time quantitative PCR.Results:(1)High purity of hUC-MSCs were successfully harvested and passaged.And there was no significant difference in the morphology of hUC-MSCs in the experimental group compared to the control group;(2)The expression of CD54 and CD106 on hUC-MSCs in the experi⁃mental group were significantly increased compared to the control group;(3)There was no difference in adipogenic dif⁃ferentiation of hUC-MSCs between 2 groups,while the calcium deposition which indicated osteogenic differentiation was less in the experimental groups than that in the control group;(4)The proliferation ability of hUC-MSCs in the experi⁃mental group was weaker than that in the control group;(5)When hUC-MSCs were co-cultured with PB

关 键 词：肿瘤坏死因子Α 脐带间充质干细胞 

分 类 号：R392.12[医药卫生—免疫学]