摘要
目的探讨瑞马唑仑(Rem)联合七氟醚(Sev)诱导肿瘤相关巨噬细胞(TAM)极化对胃癌细胞转移的调控作用及机制。方法在THP-1细胞中加入终浓度为320 ng/mL的佛波酯刺激12 h为M0型TAM(M0-TAM),然后,加入佛波酯(100 ng/mL)+IL-4(20 ng/mL)+IL-13(20 ng/mL)刺激48 h,诱导为M2型TAM(M2-TAM)。将人胃癌HGC-27细胞随机分为对照组、Rem组、Sev组和Rem+Sev组。收集各组HGC-27细胞,利用Transwell共培养体系,分别与M0-TAM或M2-TAM建立上下双层细胞共培养体系。采用集落形成实验检测细胞增殖,Transwell实验检测细胞迁移和侵袭;流式细胞术检测巨噬细胞极化;ELISA法检测共培养体系上清液中转化生长因子-β(TGF-β)、肿瘤坏死因子(TNF)-α、白细胞介素(IL)-6和IL-1β含量;Western blot检测各组共培养体系巨噬细胞极化因子精氨酸酶(Arg)-1和诱导型一氧化氮合酶(iNOS),HGC-27细胞中上皮-间充质转化(EMT)相关蛋白E-钙粘连蛋白(E-cadherin)、波形蛋白(Vimentin)、锌指结构E-box-结合同源框1(ZEB1)、扭曲因子(Twist)以及smad通路相关蛋白磷酸化SMDA家族成员2(p-smad2)、磷酸化SMDA家族成员3(p-smad3)和SMDA家族成员2/3(smad2/3)的表达。结果与Sev组相比,Rem+Sev组胃癌细胞的集落形成数和迁移、侵袭细胞数显著降低(P<0.05)。在M0-TAM和HGC-27共培养体系中,与Sev组相比,Rem+Sev组巨噬细胞中Arg-1表达和上清液中IL-10含量降低(P<0.05),iNOS表达、TNF-α和IL-1β含量增多(P<0.05)。在M2-TAM与HGC-27共培养体系中,与Sev组比较,Rem+Sev组HGC-27细胞集落形成数和迁移、侵袭细胞数减少(P<0.05),E-cadherin表达升高(P<0.05),Vimentin、ZEB1和Twist表达降低(P<0.05),TGF-β含量及p-smad2和p-smad3表达升高(P<0.05)。结论Rem联合Sev可能通过促使TAM向M2型极化,抑制胃癌细胞中TGF-β/smad信号通路介导的EMT进程,从而控制胃癌细胞的增殖、侵袭和转移。
Objective To explore the regulatory effect and mechanism of polarization of tumor-associated macrophages(TAM)induced by Remimazolam(Rem)combined with Sevoflurane(Sev)on the metastasis of gastric cancer cells.Methods THP-1 cells were treated with phorbol 12-myristate 13-acetate(PMA)at a final concentration of 320 ng/mL for 12 h to induce M0-type TAMs(M0-TAMs).Subsequently,cells were stimulated with PMA(100 ng/mL),interleukin-4(IL-4,20 ng/mL),and interleukin-13(IL-13,20 ng/mL)for 48 h to induce M2-type TAMs(M2-TAMs).Human gastric cancer HGC-27 cells were randomly assigned to control group,Rem group,Sev group,and Rem+Sev group.Cells from each group were collected and co-cultured with either M0-TAMs or M2-TAMs using a Transwell co-culture system.Cell proliferation was assessed by colony formation assay,and cell migration and invasion were evaluated using Transwell assays.Macrophage polarization was analyzed by flow cytometry.Levels of transforming growth factor-β(TGF-β),tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),and IL-1βin the co-culture supernatant were quantified by enzyme-linked immunosorbent assay(ELISA).Western blotting was employed to detect the expressions of macrophage polarization markers arginase-1(Arg-1)and inducible nitric oxide synthase(iNOS),epithelial-mesenchymal transition(EMT)-related proteins,including E-cadherin,Vimentin,zinc finger E-box-binding homeobox 1(ZEB1),Twist,and smad pathway-related proteins phosphorylated smad2(psmad2),phosphorylated smad3(p-smad3),and total smad2/3.Results Compared with Sev group,colony formation,migration,and invasion of gastric cancer cells significantly reduced in Rem+Sev group(P<0.05).In the M0-TAM and HGC-27 co-culture system,compared with Sev group,Arg-1 expression in macrophages and IL-10 levels in the supernatant were decreased in Rem+Sev group(P<0.05),while iNOS expression and TNF-α and IL-1β levels were increased(P<0.05).In the M2-TAM and HGC-27 co-culture system,compared with Sev group,the number of colony formation and the migration and invasion of HGC-27 cells were decreased in Rem+Sev group(P<0.05),the expression of E-cadherin was elevated(P<0.05),and the expressions of Vimentin,ZEB1 and Twist were reduced(P<0.05),and TGF-β content and p-smad2 and p-smad3 expressions were elevated(P<0.05).Conclusion Rem combined with Sev may inhibit TGF-β/smad pathway-mediated EMT in gastric cancer cells by modulating TAM polarization,thereby suppressing the proliferation,invasion,and metastasis of gastric cancer cells.
作者
赵昆
马宾
谢金兰
ZHAO Kun;MA Bin;XIE Jinlan(Department of Anesthesiology,Jinan People's Hospital,Affiliated People's Hospital of Shandong First Medical University,Jinan 271199,China)
出处
《山西医科大学学报》
2025年第7期736-744,共9页
Journal of Shanxi Medical University
基金
山东省医药科技项目(202205031071)。
