【摘要】 目的:探究过表达肿瘤坏死因子相关凋亡诱导配体(TRAIL)的小鼠骨髓间充质干细胞(BMSCs)对肺癌A549细胞系生物学功能的影响。方法:通过慢病毒载体携带TRAIL感染小鼠BMSCs,构建过表达TRAIL的小鼠BMSCs,RT-PCR法检测BMSCs中TRAIL基因表达。根据是否感染TRAIL基因,将细胞分为BMSCs组、NC-BMSCs组和TRAIL-BMSCs组,收集各组BMSCs的培养基,作为A549细胞条件培养基。根据条件培养基的不同,将A549细胞分为A549组、A549-BMSCs组、A549-NC-BMSCs组和A549-TRAIL-BMSCs组。MTT、Transwell和平板克隆形成实验检测各组A549细胞的增殖、迁移、侵袭和克隆能力,Westernblot法检测细胞中E-cadherin、N-cadherin和Vimentin的表达情况。结果:RT-PCR法检测结果显示,与BMSCs组比较,TRAIL-BMSCs组细胞中TRAIL mRNA的表达水平显著升高,过表达TRAIL小鼠BMSCs构建成功。MTT、Transwell和平板克隆形成实验检测结果显示,与A549组比较,A549-BMSCs组和A549-NC-BMSCs组细胞的增殖、迁移、侵袭和克隆能力显著增加,而A549-TRAIL-BMSCs组细胞在不同时间点的增殖能力显著降低,发生侵袭、迁移的细胞数量和克隆形成数量均显著降低;Western blot法检测结果显示,A549组、A549-BMSCs组、A549-NC-BMSCs组和A549-TRAIL-BMSCs组A549细胞中E-cadherin的表达水平分别为(0.45±0.03)、(0.23±0.03)、(0.25±0.02)、(0.95±0.09);N-cadherin的表达水平分别为(0.57±0.07)、(0.97±0.08)、(0.99±0.12)、(0.19±0.03);Vimentin的表达水平分别为(0.52±0.05)、(1.02±0.09)、(0.99±0.11)、(0.20±0.02),与A549组比较,A549-BMSCs组和A549-NC-BMSCs组细胞中E-cadherin的表达水平显著降低,N-cadherin和Vimentin的表达水平显著增加,A549-TRAIL-BMSCs组细胞中E-cadherin的表达水平显著增加,N-cadherin和Vimentin的表达水平显著降低,差异均有统计学意义(P<0.05)。结论:BMSCs可促进A549细胞的增殖、迁移、侵袭、克隆和EMT转化,BMSCs过表达TRAIL基因则对A549细胞的生物学功能具有明显抑制作用。更多还原

【Abstract】 Objective:To investigate the effect of overexpressing TRAIL mouse bone marrow mesenchymal stem cells(BMSCs) on the biological function of lung cancer A549 cell line.Methods:Mouse BMSCs were infected with TRAIL by lentiviral vector to construct mouse BMSCs overexpressing TRAIL.RT-PCR method was used to detect TRAIL gene expression in BMSCs.The cells were divided into BMSCs group,NC-BMSCs group and TRAIL-BMSCs group according to whether they were infected with TRAIL gene.The culture medium of each group of BMSCs was collected as conditioned medium for A549 cells.According to different conditioned media,A549 cells were divided into A549 group,A549-BMSCs group,A549-NC-BMSCs group and A549-TRAIL-BMSCs group.MTT,Transwell and plate clone formation experiments were used to detect the proliferation,migration,invasion and cloning ability of A549 cells in each group.Western blot was used to detect the expression of E-cadherin,N-cadherin and Vimentin in cells.Results:The results of RT-PCR showed that compared with BMSCs group,the expression level of TRAIL mRNA in cells of TRAIL-BMSCs group was significantly increased,which proved that BMSCs overexpressing TRAIL were successfully constructed.The results of MTT,Transwell and plate clonogenesis experiments showed that compared with A549 group,the proliferation,migration,invasion and clone ability of A549-BMSCs group and A549-NC-BMSCs group increased significantly,while the proliferation ability of A549-TRAIL-BMSCs group decreased significantly at different times,and the number of invasion and migration cells and clone formation decreased significantly.Western blot results showed that the expression levels of E-cadherin in A549 cells of A549 group,A549-BMSCs group, A549-NC-BMSCs group and A549-TRAIL-BMSCs group were(0.45±0.03),(0.23±0.03),(0.25±0.02),(0.95±0.09).N-cadherin expression levels were(0.57±0.07),(0.97±0.08),(0.99±0.12),(0.19±0.03).Vimentin expression levels were(0.52±0.05),(1.02±0.09),(0.99±0.11),(0.20±0.02).Compared with the A549 group,the expression levels of E-cadherin in cells of A549-BMSCs group and A549-NC-BMSCs group were significantly reduced,and the expression levels of N-cadherin and Vimentin were significantly increased.However,the expression levels of E-cadherin in cells of A549-TRAIL-BMSCs group increased significantly,while the expression levels of N-cadherin and Vimentin decreased significantly,and the differences were statistically significant(P<0.05).Conclusion:BMSCs can promote the proliferation,migration,invasion,cloning and EMT transformation of A549 cells.Overexpression of TRAIL gene in BMSCs can significantly inhibit the biological function of A549 cells.更多还原