hMSCs来源的外泌体对单核细胞增殖和分泌炎性因子的影响

Exosomes secreted by human mesenchymal stem cells inhibit the proliferation and secretion of monocytes

目的研究人间充质干细胞(hMSCs)来源的外泌体对单核细胞增殖和分泌炎性因子的影响,初步探讨外泌体的免疫调节功能。方法离心法培养hMSCs,收集培养的hMSCs上清液,利用试剂盒提取外泌体,电镜下观察其结构,Western blot检测其CD63和CD81的表达水平。密度梯度离心法分离外周血单核细胞,在培养的单核细胞中加入外泌体,MTT法检测单核细胞的增殖情况,流式细胞仪检测其凋亡情况,ELISA法检测上清液中肿瘤坏死因子-α(TNF-α)、白介素-1β(IL-1β)、白介素-6(IL-6)及白介素-8(IL-8)的分泌量。结果 hMSCs来源的外泌体在透射电镜下呈小球状,Western blot显示外泌体CD63和CD81表达阳性。MTT检测显示,在LPS的刺激下,单核细胞增殖增加;加入外泌体后单核细胞的增殖受到抑制。流式细胞仪检测结果表明外泌体具有促进单核细胞凋亡的作用。ELISA检测结果表明,外泌体能够抑制炎性因子TNF-α、IL-1β、IL-6及IL-8的分泌。结论 h MSCs来源的exosmes能够抑制单核细胞的增殖并促进其凋亡,同时抑制了单核细胞分泌炎性因子TNF-α、IL-1β、IL-6及IL-8。初步证明了h MSCs来源的外泌体具有免疫调节功能。

Objective To investigate the effect of exosomes secreted by human mesenchymal stem cells(hMSCs) on the proliferation and secretion of monocytes.Methods hMSCs were cultured by centrifugation method, and the supernatant of hMSCs was collected. Exosomes were extracted by kit and observed by SEM. CD63 and CD81 levels were tested by Western blot.Monocytes were isolated by density gradient centrifugation. Lipopolysaccharide(LPS) was used as a stimulant to induce proliferation and the synthesis of inflammatory cytokines. After treated by exosomes, MTT viability assay and apoptosis analysis by flow cytometry(FCM) were used to study the proliferative ability. Tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),interleukin-6(IL-6) and interleukin-8(IL-8) in the culture supernatant were measured by enzyme-linked immunosorbent assay(ELISA).Results Exosomes secreted by hMSCs were small ball-shaped under SEM. CD63 and CD81 were positively expressed by Western blot. MTT showed that LPS could promote the proliferation of monocytes, while exosomes inhibited the proliferation of monocytes. However, after treated by exosomes, the apoptosis of monocytes significantly increased. ELISA showed that exosomes could inhibit the secretion of TNF-α, IL-1β, IL-6 and IL-8 by monocytes.Conclusion Exosomes secreted by hMSCs can inhibit the proliferation of monocytes and the secretion of TNF-α, IL-1β, IL-6 and IL-8 by monocytes while promote the apoptosis. It is preliminarily proved that hMSC-derived exosomes have immune-regulatory function.