人肺腺癌A549干样细胞富集及其自噬表达水平研究

目的肿瘤干细胞一直都是肿瘤研究领域的热点,但其分离鉴定方法尚未成熟,关于肿瘤干细胞生物特性与自噬表达相关性研究较少。因此,本研究通过无血清悬浮培养法富集肺腺癌干样细胞,鉴定其生物特性并初步检测其自噬表达水平。方法将人肺腺癌A549亲本细胞(A549monolayer,A549-MN)作为对照组,A549干样细胞(A549stem-like cells,A549-SC)作为实验组。其中,A549-SC通过将A549-MN置于含有诱导因子重组人表皮生长因子(epidermal growth factor,EGF)、重组人碱性成纤维生长因子(basic fibroblast growth factor,bFGF)和B27的无血清悬浮培养基中富集得到。采用流式细胞术检测A549-MN及A549-SC的"CD44+"和"CD133+"标记细胞比例,分化实验检测其分化能力;Transwell迁移和侵袭实验检测其迁移侵袭能力,实时荧光定量PCR及蛋白质印迹实验检测肿瘤干细胞干性基因OCT4、Nanog、Bmi 1和自噬相关因子Beclin1、LC3B的mRNA及蛋白表达水平,透射电子显微镜观察分析A549-MN及A549-SC自噬小体形成情况。结果无血清悬浮培养法可从A549-MN中富集干样细胞且能稳定传代;A549-SC中"CD44+"及"CD133+"标记细胞比例(14.2%,7 111/50 231)高于A549-MN(1.2%,680/54 858)。分化实验表明,A549-SC在含血清的培养基中又可分化成形态学上与A549-MN无差异的贴壁细胞。Transwell实验示,A549-MN与A549-SC的迁移细胞数分别为(96±3)及(121±2)个/视野,t=9.78,P<0.01;侵袭细胞数分别为(22±1)及(52±3)个/视野,t=11.72,P<0.01,差异均有统计学意义。与A549-MN相比,A549-SC的干性基因OCT4 (t=2.41,P<0.05)、Nanog (t=13.84,P<0.01)、Bmi 1(t=14.56,P<0.01)及自噬因子Beclin 1(t=7.44,P<0.01)、LC3B (t=10.59,P<0.01)的mRNA表达水平均升高,干性基因Nanog (t=11.55,P<0.01)、Bmi 1(t=4.37,P<0.01)及自噬因子Beclin1(t=9.69,P<0.01)、LC3B (t=15.7,P<0.01)的蛋白表达水平也升高。同时,电镜观察显示A549-SC的自噬小体较A549-MN增多。结论应用无血清悬浮培养法可富集得到人肺腺癌A549干样细胞,其具有干细胞生物特性且存在高水平自噬表达现象。

Explore the enrichment and autophagy level of human lung adenocarcinoma cell line A549 stem-like cells

OBJECTIVE Cancer stem cells are hotspots in the field of cancer research,but their isolation and identification methods are not yet mature.What’s more,few studies explored the biological characteristics and autophagy levels of cancer stem-liked cells.Therefore,this study aimed to explore the enrichment of lung cancer stem-like cells through serum-free suspension culture and detect their level of autophagy expression.METHODS Human lung adenocarcinoma cell line A549 monolayer cells(A549-MN)were used as control group,and A549 stem-liked cells(A549-SC)were used as experimental group.A549-MN was suspended in serum free medium containing inducible factors EGF,bFGF,B27 to form A549-SC.Then we used fluorescence activated cell sorter to detect the percent of"CD44+"and "CD133+"cells in A549-MN and A549-SC;Differentiation assay to detect their differentiation ability;Transwell migration and invasion assays to detect their migration and invasion abilities;qRT-PCR and Western Blotting to detect genes and proteins expression of stemness factors OCT4,Nanog,Bmi 1 and autophagy markers Beclin1,LC3 B;Electron transmission microscopy to observe the number of autophagosome in A549-MN and A549-SC.RESULTS Serum-free suspension culture could enrich A549-SC which could be passaged stably;The percent of"CD44+"and"CD133+"cells in A549-SC was 14.2%(7 111/50 231),which was higher than 1.2%(680/54 858)in A549-MN;Differentiation assay showed that A549-SC in serum-containing medium could differentiate into adherent cells that were morphologically indistinguishable from A549-MN.Transwell experiments showed that the number of migration cells of A549-MN and A549-SC were(96±3)/field of view and(121±2)/field of view respectively,t=9.78,P<0.01;the number of invasion cells were(22±1)/field of view and(52±3)/field of view respectively,t=11.72,P<0.01,and the differences were both statistically significant.Compared with A549-MN,the mRNA levels of stemness,OCT4(t=2.41,P<0.05),Nanog(t=13.84,P<0.01),Bmi1(t=14.56,P<0.01),and autophagic factors,Beclin 1(t=7.44,P<0.01),LC3 B(t=10.59,P<0.01),of A549-SC were increased.what’s more,proteins expression levels of stemness,Nanog(t=11.55,P<0.01),Bmi 1(t=4.37,P<0.01),and autophagic factors,Beclin1(t=9.69,P<0.01),LC3 B(t=15.7,P<0.01)showed the same change.Meanwhile,electron microscopy showed that the autophagosome of A549-SC increased compared with A549-MN.CONCLUSION Human lung adenocarcinoma A549 stem-like cells can be enriched by serum-free suspension culture,which has stem cell characteristics and high levels of autophagy expression.