Toll样受体预活化的人脂肪间充质干细胞对人肺癌H460细胞生长的抑制作用

Inhibitory effect of human adipose-derived stem cells pre-activated with Toll-like receptor on growth of human lung cancer H460 cells

目的观察聚肌胞苷酸[polyinosinic-polycytidylic acid,简称为Poly(I∶C)]和脂多糖(lipopolysaccharide,LPS)预活化后的人脂肪间充质干细胞(human adipose-derived stem cells,hADMSCs)对人肺癌H460细胞生长的抑制作用。方法利用抽脂手术遗弃脂肪组织标本制备hADMSCs,并鉴定其细胞表面分子标志、细胞增殖能力和体外分化能力;比较Poly(I∶C)、LPS预活化后的hADMSCs细胞表面标志和体外分化能力;ELISA法测定Poly(I∶C)、LPS预活化后的hADMSCs培养基上清中IFNβ的分泌情况;Western blot法分析Poly(I∶C)预活化后的hADMSCs TRAIL的表达情况;用不同浓度Poly(I∶C)预活化hADMSCs后,与不同比例的H460共培养,分析对肿瘤细胞的抑制作用。结果光学显微镜下hADMSCs呈纤维样、鱼群样排列;hADMSCs细胞表面标志CD44、CD73、CD90、CD105、HLA-ABC的阳性率均大于95%,造血干细胞的特异性标志物CD34、CD45的阴性率均大于98%;Poly(I∶C)和LPS刺激的hADMSCs其细胞表面标志未受影响。Poly(I∶C)和LPS能促进hADMSCs的成骨分化,对成脂分化能力无明显作用。经Poly(I∶C)诱导的hADMSCs分泌的IFNβ浓度明显高于未经诱导的对照组(P<0.05),低浓度的LPS诱导hADMSCs IFNβ表达量不高。Poly(I∶C)预处理促进了hADMSCs表达TRAIL。体外5和25μg/m L的Poly(I∶C)预活化后的hADMSCs对H460细胞表现出细胞毒性,对H460细胞的生长有抑制作用;hADMSCs∶H460比例为1∶2时,对H460细胞的抑制效果最佳。结论成功分离、培养出hADMSCs,其在体外低浓度的Poly(I∶C)诱导下能表达TRAIL,并对H460细胞的生长产生抑制作用。

Objective To observe the inhibitory effect of human adipose-derived mesenchymal stem cells(hADMSCs)pre-activated with polyinosinic-polycytidylic acid[Poly(I∶C)]and lipopolysaccharide(LPS)on growth of human lung cancer H460 cells.Methods The hADMSCs were isolated from adipose tissue samples of patients receiving liposuction and identified for cell surface molecular markers,cell proliferation ability and differentiation ability in vitro.The cell surface molecular markers and differentiation ability in vitro of hADMSCs pre-activated with Poly(I∶C)and with LPS were compared.The secretion of IFNβin culture supernatant of hADMSCs pre-activated with Poly(I∶C)and LPS were determined by ELISA,while the expression of TRAIL in hADMSCs pre-activated with Poly(I∶C)by Western blot.The hADMSCs pre-activated with various concentrations of Poly(I∶C)were co-cultured with H460 cells at various ratios,of which the inhibitory effect on tumor cells was analyzed.Results The hADMSCs under optical microscope were in fiberlike and fish-like arrangements.All the positive rates of surface markers CD44,CD73,CD90,CD105 and HLA-ABC on hADMSCs were more than 95%,while all the negative rates of specific surface markers CD34 and CD45 of hematopoietic stem cells were more than 98%.The stimulation with Poly(I∶C)and LPS showed no influence on the surface markers on h ADMSCs.Both Poly(I∶C)and LPS promoted the osteogenic differentiation of hADMSCs while showed no significant effect on the adipogenic differentiation.The IFNβconcentration in hADMSCs induced with Poly(I∶C)was significantly higher than that without induction(P<0.05).However,the expression level of IFNβin HADMSCs induced with low concentration LPS was not high.The pre-treatment with Poly(I∶C)promoted the expression of TRAIL in hADMSCs.The h ADMSCs pre-activated with 5 and 25μg/mL showed cytotoxicity to H460 cells,while showed inhibitory effect on the growth of H460 cells.The mixture of hAMSCs and H460 cells at a ratio of 1∶2 showed satisfactory inhibitory effect on H460 cells.Conclusion The hADMSCs were successfully isolated and cultured,in which TRAIL was expressed under induction of low concentration Poly(I∶C).However,the induction inhibited the growth of H460 cells.