摘要
目的:研究肠上皮细胞(intestinal epithelial cells,IEC)来源的整合素αVβ6对骨髓来源树突状细胞(bone marrow-derived dendritic cells,BMDCs)功能的影响.方法:分离、培养小鼠IEC和DC,IEC经卵清蛋白(ovalbumin,OVA)刺激后,多步离心法制备外泌体(exosome).应用免疫胶体金对外泌体中的整合素αVβ6进行定性,通过免疫磁珠技术,分离获取DC,流式细胞仪检测分离后DC纯度.然后将分离后的DC分5组,分别为空白组、OVA组、外泌体组、外泌体+抗整合素αVβ6抗体组、外泌体+羊抗小鼠IgG组,观察各组对脂多糖(lipopolysaccharide,LPS)刺激的反应.ELISA法检测DC在LPS刺激前后IL-12p70的表达.LPS刺激前活性转化生长因子β1(transforming growth factor-β1,TGF-β1)以及总TGF-β1的表达.结果:OVA组与空白组相比,总TGF-β1表达明显增高(226.636±40.355vs176.947±23.072,P<0.05),但活化TGF-β1表达无明显变化(P>0.05).空白组与外泌体+抗整合素αVβ6抗体组相比,活化TGF-β1表达无明显变化(P>0.05),但总TGF-β1表达明显增高(P<0.05).外泌体组和外泌体+羊抗小鼠IgG组与空白组相比,活化TGF-β1和总TGF-β1表达均明显增高(P<0.05).相比空白对照组,LPS刺激48h后外泌体组和外泌体+羊抗小鼠IgG组IL-12p70表达明显降低(P<0.05),OVA组及外泌体+抗整合素αVβ6抗体组IL-12p70表达无明显差异(P>0.05).结论:IEC来源整合素αVβ6能增加DC活性,以及TGF-β1和总TGF-β1的表达量,并拮抗LPS对BMDC的促成熟作用.
AIM：To investigate the effect of intestinal epithelial cell（IEC）-derived integrin αVβ6 on the biological characteristics of bone marrow-derived dendritic cells（BMDCs）.METHODS：IECs and BMDCs were separated from BALB/c mice and cultured.After IECs were stimulated with ovalbumin（OVA）,exosomes were prepared by multiple-step centrifugation.The expression of integrin αVβ6 in exosomes was examined by using the immune colloidal gold technique.Dendritic cells（DCs） were separated using immunomagnetic beads,and the concentration of DCs was determined by flow cytometry.DCs were then divided into five groups：blank group,OVA group,exosomes group,exosomes plus anti-αVβ6 antibody group,and exosomes plus goat anti-mouse IgG group.After these groups of DCs were treated with LPS,the expression of IL-12p70 was detected.In addition,the expression of active and total TGF-β1 was detected before LPS stimulation.RESULTS：Compared to the blank group,the expression levels of total TGF-α1 increased（both P 〈 0.05） and those of active TGF-β1 showed no significant changes（both P 〉 0.05） in the OVA group and exosomes plus anti-αVβ6 antibody group;and the expression levels of both active and total TGF-β1 increased in the exosomes group and exosomes plus goat anti-mouse IgG group（both P 〈 0.05）.Compared to the blank group,the expression of IL-12p70 was significantly reduced（both P 〈 0.05） in the exosomes group and exosomes plus goat anti-mouse IgG group,but showed no signif icant changes in the OVA group and exosomes plus anti-αVβ6 antibody group（both P 〉 0.05） 48 h after stimulation with LPS.CONCLUSION：Intestinal epithelial cell-derived integrin αVβ6 can increase the expression of active TGF-β1 and total TGF-β1 in DCs and antagonize LPS-induced BMDC maturation.
出处
《世界华人消化杂志》
CAS
北大核心
2012年第17期1553-1558,共6页
World Chinese Journal of Digestology
基金
国家自然科学基金资助项目
No.31070799~~
