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hp-MSCs对RSV引起小儿毛细支气管炎的保护作用及机制研究
李得志,于欣欣,吴福玲,高萌,石涛
0
(滨州医学院附属医院儿科,山东 滨州 256610;滨州市人民医院儿科,山东 滨州 256610)
摘要:
目的 探讨人胎盘间充质干细胞(hp-MSCs)水平降低与毛细支气管炎发病的相关性及hP-MSCs防治毛细支气管炎的分子机制。方法 在滨州医学院附属医院产科招募志愿者,同时收集新生儿胎盘。毛细支气管炎患者由儿科医生根据临床诊断标准做出诊断;以毛细支气管炎患者为病例组,正常体检儿童为对照组,进行病例对照研究;胎盘hp-MSCs水平检测采用锥虫蓝染色计数法,hp-MSCs表面标记物的检测采用流式细胞仪方法;采用ELISA(酶联免疫吸附法)检测白细胞介素-9(IL-9)、白细胞介素-17(IL-17)、白细胞介素10(IL-10)和转化生长因子-1(TGFβ-1)水平,采用Real time PCR和Western blot法检测叉头框蛋白3(Foxp3)及维甲酸相关孤核受体γt(RORγt)基因和蛋白表达水平。结果 两组人群在是否存在特异性皮炎、毛细支气管炎1级和2级家族史有显著性差异(P<0.05)。病例组hp-MSCs水平及其表面标记物CD44和CD29水平显著降低(P<0.05)。呼吸道合胞体病毒(RSV)感染升高IL-17和IL-9等炎症因子水平,降低IL-10和TGFβ-1等抗炎因子水平;而hp-MSCs干预可以降低IL-17和IL-9水平而升高IL-10和TGFβ-1水平(均P<0.05)。RSV感染可以下调Foxp3基因和蛋白表达,上调RORγt基因和蛋白表达;而hpMSCs干预可以上调Foxp3基因和蛋白表达,下调RORγt基因和蛋白表达。结论 胎盘hpMSCs水平降低是引起婴幼儿毛细支气管炎的独立危险因素,hp-MSCs通过上调Foxp3的蛋白和基因表达,以及下调RORγt蛋白和基因表达起到防治婴幼儿毛细支气管炎的作用。
关键词:  人胎盘间充质干细胞  毛细支气管炎  辅助性T细胞17 /调节性T细胞  叉头框蛋白3  维甲酸相关孤核受体γt
DOI:
基金项目:山东省自然科学基金(ZR2014HL001)
The effection and mechanism of human placenta derived mesenchymal stem cell on the capillary bronchitis caused by respiratory syncytial virus
LI Dezhi,YU Xinin,WU Fuling,GAO Meng,HI Tao
(Department of Paediatrics, Binzhou Medical University Hospital, Binzhou 256610, Shandong,China;Department of Paediatrics, Binzhou People’s Hospital, Binzhou 256610, Shandong, China)
Abstract:
Objective To investgate the relationship between bronchiolitis in infant and the decrease in -PMSCs and the mechanism of hp-MSCs in the prevention and treatment of bronchiolitis.Methods Volunteers were recruited according to the inclusion and exclusion criteria. Patients with bronchiolitis were diagnosed by a pediatrician. At the same time, infant placenta was collected. Cell growth was detected by trypan blue exclusion. HpMSCs surface markers were detected by flow cytometry. Separation of peripheral blood mononuclear cells from peripheral blood was operated by density gradient centrifugation with HypaqueFicoll. CD4+T lymphocytes were further isolated by MACS from mononuclear cells. I-9, IL-17, IL-10 and TGF beta1 levels were detected using ELISA method. IL-9, IL-17, IL-10 and TGF beta1 levels were detected using ELISA method. the expression of the gene and protein in Foxp3 and RORγ were respectively measured by wetern blot and Real time PCR. Results It is significant different between two group in the specific dermatitis. The hp-MSCs level in control was significant higher than that in case group. The expression of CD44 and CD29 Lymphocyte, placental hpMSC surface marker in the control group were significantly higher than that in case group. IL17 and IL-9 from CD4+ T lymphocytes were increased by RSV() infection, Compared with that of the control group. The increase in IL-17 and IL-9 level caused by RSV infection was reduced with hpMSCs intervention. Compared with the control group, the levels of CD4+T lymphocytes secreting IL-10 and TGF,1 were significantly reduced in RSV group, and IL10 and TGF,1 level were significantly enhanced in hp-MSCs group. Compared with the control group, the expression of Foxp3 gene and protein were downregulated by RSV infection, the expression of Foxp3 gene and protein were upregulated by hp-MSCs intervention. Compared with the control group, the expression of RORγt gene and protein were upregulated by RSV infection, the expression of RORγt gene and protein were downregulated by hp-MSCs intervention.Conclusion Reduction of placental hp-MSCs levels is an independent risk factor for infant bronchiolitis. The balance of TH17/Treg is managed by hpMSCs through upregulation of Foxp3 protein and gene expression,downregulation of RORγt protein and gene expression as well, it is further raised the increase of antiinflammatory cytokine secretion,and decrease of inflammatory factor secretion.
Key words:  hp-MSC  Bronchiolitis  TH17/Treg  Foxp3  RORγt

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