【作者】 张丹；

【导师】 何大维；

【作者基本信息】 重庆医科大学 ， 儿科学， 2019， 硕士

【摘要】 目的:探讨Piwil2诱导肿瘤干细胞(Piwil2 induced cancer stem cells,Piwil2-iCSC)来源的外泌体对人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hucMSCs)肿瘤样生物学行为的影响。方法:采用超速离心法提取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(Control)、PBS干预组(PBS)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50~100nm,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hucMSCs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-iCSC外泌体干预后的hucMSCs细胞MMP2(P<0.05 vs PBS组,P<0.01 vs Control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。更多还原

【Abstract】 Objective:To investigate whether Piwil2-iCSCs derived exosomes could have an effect on the proliferation,migration and invasion of human umbilical cord mesenchymal stem cell(hucMSCs).Methods: Piwil2-iCSCs derived exosomes were isolated by ultracentrifugation,then identified by transmission electron microscopy,Nanoparticle Tracking Analysis and western-blot.Exosome uptake assay was used to detect the pathway how the Piwil2-iCSCs derived exosomes works.HucMSCs were divided into control group(Control),PBS intervention group(PBS)and exosome intervention group(Exo).CCK-8,Wound healing assay,Transwell assay,western-blot and cell karyotype analysis were used to detecting the proliferation,migration,invasion,the expression levels of MMP2 and MMP9 proteins,and chromosome structure of hucMSCs.Results: The diameter of Piwil2-iCSCs derived exosomes was about 50nm~100nm,most of them were oval or spherical and coated with membrane and rich in CD9,CD63 and Piwil2 proteins.Exosomal uptake assay indicated that exosomes entered the cell to work.Compared with the Control group and the PBS group,the number of cell proliferation in the Exo group was significantly increased(P<0.05);the healing rate of scratches was accelerated(24h,P<0.05;48h,P<0.01);the number of invasive cells was significantly increased(P<0.01);the expression of MMP2(P<0.05 vs PBS group,P<0.01 vs Control group)and MMP9(P<0.05)protein levels in hucMSCs cells treated with Piwil2-iCSC exosomes increased;but the karyotype was still 46 XY with no abnormalities.Conclusion: Piwil2-iCSCs derived exosomes could promote the proliferation,migration and invasion of hucMSCs,and no cancer-like heterogeneity changes.更多还原