肿瘤样干细胞来源的外泌体促进人脐带间充质干细胞的增殖和侵袭

Cancer stem-like cell-derived exosomes promotes the proliferation and invasion of human umbilical cord blood-derived mesenchymal stem cells

目的探讨Piwil2诱导肿瘤干细胞(Piwil2-iCSC)来源的外泌体对人脐带间充质干细胞(hucMSCs)肿瘤样生物学行为的影响。方法超速离心法提取Piwil2-iCSCs外泌体,透射电镜、粒径分析、Western blot及外泌体摄入实验检测外泌体形态、直径、标志蛋白表达及作用途径。将hucMSCs分为对照组、PBS干预组和外泌体干预组(Exo),CCK-8、细胞划痕实验、Transwell和Western blot检测各组hucMSCs的增殖、迁移、侵袭能力以及基质金属蛋白酶(MMPs)MMP2和MMP9蛋白的表达水平。细胞染色体核型分析检测经Piwil2-iCSCs外泌体长期干预的hucMSCs的染色体结构。结果透射电镜可见Piwil2-iCSCs外泌体大小均匀,直径在50～100 nm,呈形态较规则的圆形或椭圆形囊泡状小体,粒径分析显示外泌体直径分布集中在在100～200 nm;Western blot显示外泌体标志性蛋白CD9、CD63和piwil2蛋白阳性表达;外泌体摄入实验表明外泌体进入细胞发挥作用。与对照组和PBS干预组相比,Exo组细胞增殖数量明显增多(P<0.05),划痕愈合速度加快(24 h,P<0.05;48 h,P<0.01),侵袭细胞数目显著增加(P<0.01)。经Piwil2-iCSCs外泌体干预后的hucMSCs细胞MMP2(P<0.05 vs PBS干预组,P<0.01 vs对照组)、MMP9蛋白水平表达增高(P<0.05),但染色体核型仍是46XY,无异常。结论肿瘤样干细胞Piwil2-iCSC外泌体能促进hucMSCs的增殖、迁移及侵袭,但是未出现肿瘤样异质性改变。

Objective To investigate the effect of Piwil2-induced cancer stem-like cell (Piwil2-iCSC)-derived exosomes on the proliferation, migration and invasion of human umbilical cord blood-derived mesenchymal stem cells (hucMSCs). Methods Piwil2-iCSC-derived exosomes were isolated by ultracentrifugation and identified using transmission electron microscopy, nanoparticle tracking analysis and Western blotting. Exosome uptake assay was used to identify the pathway that Piwil2-iCSCderived exosomes utilized. HucMSCs were divided into control group, PBS intervention group and exosome intervention group, and CCK-8 assay, wound healing assay, Transwell assay, Western blotting and cell karyotype analysis were used to observe the proliferation, migration, invasion, expression levels of MMP2 and MMP9 proteins, and chromosome structure of hucMSCs. Results The diameter of Piwil2-iCSC-derived exosomes ranged from 50 nm to 100 nm, and most of them were oval or spherical capsules rich in CD9, CD63 and Piwil2 proteins. Exosomal uptake assay showed that the exosomes executed theirs functions after entering the cells. Compared with the control cells and PBS-treated cells, hucMSCs treated with the exosomes showed significantly increased number of proliferating cells (P<0.05) with accelerated healing rate (P<0.05 at 24 h; P<0.01 at 48 h), increased invasive cells (P<0.01), enhanced protein expressions of MMP2 (P<0.05 vs PBS group; P<0.01 vs control group) and MMP9 (P<0.05), but their karyotype still remained 46XY without any abnormalities. Conclusion Piwil2-iCSC-derived exosomes can promote the proliferation, migration and invasion but does not cause cancer-like heterogeneity changes in hucMSCs.