| Objective:To investigate the expression of IFN-βprotein in human umbilical Cord Derived mesenchymal stem cells transfected with interferon-βgene and to furture explore the effect of IFNβ-hucMSCs on the clone formation,migration and proliferation of non-small cell lung cancer cell lines A549 and H226.Methods:(1)Isolation,culture and amplification of primary huc-MSCs:Umbilical Cord Derived mesenchymal stem cells were isolated from neonatal umbilicl cord by tissue adherence method and analyzed by flow cytometry.(2)The huc-MSCs were infected by lentivirus-IFNβwith enhanced red fluorescent protein-labeled and take its supernatant to deal with cancer cells were divided into the following five groups:Lentivirus-IFNβtransfection hucMSCs group(IFNβ-hucMSCs group),Lentivirus empty carrier transfection hucMSCs group(NC group),Mesenchymal stem cell group(hucMSCs group),Interferon group(IFN-βgroup),Blank control group(CON group).(3)The content and expression of IFN-βprotein in three groups of IFN-βgroup,NC group and hucMSCs group was detected by ELISA and Western blot,respectively.(4)After A549 cells and H226 cells were treated with IFNβ-hucMSCs group,NC group,hucMSCs group,IFNβgroup and CON group,the clone formation rate of A549cells and H226 cells were detected by Giemsa’s staining.Transwell was used to detect the migration ability of these groups.CCK-8 assay was used to measure the proliferation ability of these groups.Results:(1)The mesenchymal stem cells were isolated from umbilicl cord by tissue adherence method.After growing for about 2 weeks,shuttle cells began to crawl out from the umbilical cord,and cells gradually fused and covered with T25cm~2 culture bottles.Cells passage to the 2-3generation can be observed in the cell colonies,forming a whirlpool,with a more consistent form.The expression of hucMSCs related surface markers CD14、CD90、CD105 of stromal cells;but the monocytes、lymphocytes and hematopoietic stem cell surface markers CD14、CD19、CD34 without;do not express human leukocyte common antigen CD45 and MHC-II class HLA-DR molecules,which is consistent with the phenotype characteristics of mesenchymal stem cells.(2)The content of IFN-βprotein in three groups of the supernatants was detcted by ELISA,the content of IFN-βprotein in the supernatant of IFNβ-hucMSCs group24h(1228.162±72.940pg/ml)、48h(1218.693±17.583pg/ml)、72h(1045.363±41.176pg/ml)at three time points was significantly higher than that in NC group24h(45.599±0.792pg/ml)、48h(47.841±2.553pg/ml)、72h(47.154±1.078pg/ml)and in hucMSCs group 24h(46.806±2.659pg/ml)、48h(41.955±3.007pg/ml)、72h(41.435±2.171pg/ml)and the difference was statistically significant(P<0.01).The expression of IFN-βprotein in three groups was detected by Western blotting,the expression level of IFN-βprotein in IFNβ-hucMSCs group was(0.684±0.136),which was significantly higher than that in NC group(0.112±0.027)and hucMSCs group(0.105±0.042),the difference was statistically significant(P<0.01).(3)After A549 cells were treated,the clone formation rates of IFNβ-hucMSCs group and IFN-βgroup were(5.557±0.306)%、(8.200±0.436)%,respectively,which were lower than that of NC group(13.567±0.289)%、hucMSCs group(13.367±0.153)%、CON group(14.000±0.200)%,and the clone formation rate of IFNβ-hucMSCs group was significantly lower than that of IFN-βgroup(P<0.01).The relative migration rates of IFNβ-hucMSCs group and IFN-βgroup were 0.563%、1.150%,respectively,which were lower than that of NC group(1.842)%、hucMSCs group(1.823)%、CON group(1.850)%,and the relative migration rate of IFNβ-hucMSCs group was lower than that of IFN-βgroup(P<0.01).The absorbance values of IFNβ-hucMSCs group and IFN-βgroup at 72h were lower than those of NC group、hucMSCs group and CON group,and IFNβ-hucMSCs group was also lower than that of IFN-βgroup,(P<0.01).(4)After H226 cells were treated,the clone formation rates of IFNβ-hucMSCs group and IFN-βgroup were(1.800±0.500)%、(3.100±0.100)%,respectively,which were lower than that of NC group(10.800±0.265)%、hucMSCs group(10.367±0.208)%、CON group(11.300±0.361)%,and the clone formation rate of IFNβ-hucMSCs group was significantly lower than that of IFN-βgroup(P<0.01).The relative migration rates of IFNβ-hucMSCs group and IFN-βgroup were 0.307%、0.588%,respectively,which were lower than that of NC group(1.115)%、hucMSCs group(1.110)%、CON group(1.140)%,and the relative migration rate of IFNβ-hucMSCs group was lower than that of IFN-βgroup(P<0.01).The absorbance values of IFNβ-hucMSCs group and IFN-βgroup at 72h were lower than those of NC group、hucMSCs group and CON group,and IFNβ-hucMSCs group was also lower than that of IFN-βgroup,(P<0.01).Conclusion:IFNβ-hucMSCs could significantly inhibit the malignant biological behaviors of non-small cell lung cancer cell lines A549 and H226,such as clone formation,migration and proliferation capabilities. |
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