肺瘤平膏含药血清对巨噬细胞共培养条件下A549细胞EMT相关mRNA及蛋白表达的影响

Effects of Feiliuping Ointment Containing Serum on EMT Related m RNA and Protein Expressions under Co-culture Conditions of A549 Cells and Macrophage

目的观察肺瘤平膏含药血清对A549细胞与巨噬细胞共培养条件下上皮间质转化(epithelialmesenchymal transition,EMT)相关mRNA及蛋白表达的影响。方法制备肺瘤平膏含药血清,建立A549细胞与巨噬细胞的共培养体系,将细胞分为A549加空白血清组(A549+NRS组)、共培养加空白血清组(共培养+NRS组)及共培养加肺瘤平膏含药血清组(共培养+FLP组)。采用荧光定量PCR、免疫荧光法比较各组A549细胞EMT相关基因(SNAIL1、SNAIL2、ZEB1、CDH1、CDH2、VIM、TJP1、CLDN1、CTNNB1、FLRT1)及E-cadherin、N-cadherin、ZO-1、Vimentin蛋白表达水平的影响。结果共培养条件下,A549细胞变为间充质细胞样。加入肺瘤平膏含药血清后,部分A549细胞形态恢复为上皮细胞样。与A549+NRS组比较,共培养+NRS组A549细胞SNAIL1、ZEB1、CTNNB1、FLRT1、CDH2、VIM mRNA表达上调,TJP1 mRNA表达下调(均P<0.05);与共培养+NRS组比较,共培养+FLP组SNAIL2、TJP1、CLDN1、CDH1、VIM mRNA表达上调,CTNNB1、FLRT1及CDH2 mRNA表达下调(均P<0.05)。免疫荧光结果显示,E-cadherin表达于细胞膜和胞浆内,以细胞膜为主;N-cadherin表达于细胞膜和胞浆内,以细胞浆为主;Vimentin表达于胞浆内;ZO-1表达于细胞连接处为主,部分细胞膜染色亦呈阳性。与A549+NRS组比较,共培养+NRS组A549细胞E-cadherin蛋白表达下调,N-cadherin及Vimentin蛋白表达上调(P<0.05);肺瘤平膏含药血清可上调E-cadherin表达,下调N-cadherin及Vimentin蛋白表达(均P<0.05)。结论肺瘤平膏可抑制共培养条件下A549细胞EMT的形成。

Objective To observe the effects of Feiliuping Ointment （FLP） containing serum on A549 cell epithelial-mesenchymal transition （EMT） related mRNA and protein expressions under macro- phage co-culture conditions. Methods FLP containing serum was prepared. A co-culture system of A549 cells and macrophages was established. A549 cells were divided into 3 groups, i.e., the blank serum group （A549 ＋NRS）, the co-culture cells ＋ blank serum group （co-culture ＋ NRS）, the co-culture cells with FLP containing serum group （co-culture ＋ FLP）. The effects of FLP on A549 cell EMT related gene （SNAIL1, SNAIL2, ZEB1, CDH1, CDH2, VIM, TJP1, CLDN1, CTNNB1, FLRT1 ） and proteins （E-cadherin, N-cadher- in, ZO-1, Vimentin） expressions were observed under co-culture conditions by fluorescent quantitative PCR. Results A549 cells developed into mesenchymal-like cells in co-culture conditions, which could been blocked by FLP containing serum in part. Compared with the A549 ＋NRS group, mRNA expressions of SNAIL1, ZEB1, CTNNB1, FLRT1, CDH2, and VIM were up-regulated （P 〈0.05）, but the expression of TJP1 was down-regulated in the co-culture ＋ NRS group （all P 〈 0.05）. Compared with the co-culture ＋ NRS group, mRNA expressions of SNAIL2, TJP1, CLDN1, CDH1, and VIM were up-regulated, but mRNA expressions of CTNNB1, FLRT1, and CDH2 were down-regulated in the co-culture ＋ FLP group （all P 〈0.05）. Immunofluorescent results showed that E-cadherin expressed on cell membrane and inside cytoplasm, and most expressed on cell membrane. N-cadherin expressed on cell membrane and inside cytoplasm, and most expressed inside cytoplasm. Vimentin expressed within the cytoplasm. ZO-1 expressed mainly in cell junction. Parts of the cell membrane were positively stained. Compared with the A549 ＋NRS group, mRNA expression of E-cadherin was down-regulated in A549 cells, and mRNA expressions of N-cadherin and Vi- mentin were up-regulated in the co-culture ＋ NRP group. However, E-cadherin was up-regulated and protein expressions of N-cadherin and Vimentin were down-regulated after intervention of FLP containing serum. （all P 〈0.05）. Conclusion FLP could inhibit the EMT of A549 cells under co-culture conditions.