BACKGROUND Inflammatory bowel disease（IBD） is characterized by chronic and non-specific inflammation of the intestinal mucosa and mainly includes ulcerative colitis and Crohn’s disease.AIM To explore the beneficial...BACKGROUND Inflammatory bowel disease（IBD） is characterized by chronic and non-specific inflammation of the intestinal mucosa and mainly includes ulcerative colitis and Crohn’s disease.AIM To explore the beneficial effect of Toxo ROP16Ⅰ/Ⅲ-induced M2 phynotype macrophages in homeostasis of IBDs through downregulation of M1 inflammatory cells.METHODS RAW264.7 macrophages stimulated by lipopolysaccharide（LPS）（M1 cells） were co-cultured with Caco-2 cells as an inflammatory model of IBD in vitro.The expression of Toxo ROP16Ⅰ/Ⅲ was observed in RAW264.7 macrophages that were transfected with p EGFP-rop16Ⅰ/Ⅲ.The phenotypes of M2 and M1 macrophage cells were assessed by quantitative real-time reverse transcriptase polymerase chain reaction and the expression of tumor necrosis factor（TNF）-α,interleukin（IL）-1β,IL-6,transforming growth factor（TGF）-β1,IL-10,inducible nitric oxide synthase（i NOS）,and arginase-1（Arg-1） was detected.The expression of i NOS,Arg-1,signal transducer and activator of transcription 3（Stat3）,p-Stat3,Stat6,pStat6,programmed death ligand-2（PD-L2）,caspase-3,-8,and-9 was analyzed by Western blotting,and Griess assays were performed to detect nitric oxide（NO）.TNF-α,IL-1β,IL-6,TGF-β1,and IL-10 expression in the supernatants was detected by enzyme-linked immunosorbent assay,and Caco-2 cell apoptosis was determined by flow cytometry after mixing M1 cells with M2 cells in a Caco-2 cell co-culture system.RESULTS M1 cells exhibited significantly increased production of i NOS,NO,TNF-α,IL-1β,and IL-6,while Toxo ROP16Ⅰ/Ⅲ induced macrophage bias to M2 cells in vitro,showing increased expression of Arg-1,IL-10 and TGF-β1 and elevated production of p-Stat3 and p-Stat6.The mixed M1 and M2 cell culture induced by Toxo ROP16 Ⅰ/Ⅲ exhibited decreased production of NO and i NOS and upregulated expression of Arg-1 and PD-L2.Accordingly,Caco-2 cells became apoptotic,and apoptosis-associated proteins such as caspase-3,-8 and-9 were dampened during co-cu展开更多
为探讨弓形虫Ⅱ型(ME49)ROP16蛋白对人乳腺癌MCF-7细胞增殖、凋亡、迁移和侵袭的影响,利用Ⅱ型ROP16(MCF-7-ROP16Ⅱ)和空载体(MCF-7-HBLV)慢病毒载体分别感染MCF-7细胞,用实时荧光PCR、Western-blot验证ROP16蛋白在MCF-7细胞的表达,用CC...为探讨弓形虫Ⅱ型(ME49)ROP16蛋白对人乳腺癌MCF-7细胞增殖、凋亡、迁移和侵袭的影响,利用Ⅱ型ROP16(MCF-7-ROP16Ⅱ)和空载体(MCF-7-HBLV)慢病毒载体分别感染MCF-7细胞,用实时荧光PCR、Western-blot验证ROP16蛋白在MCF-7细胞的表达,用CCK-8和流式细胞术检测细胞增殖和凋亡,用细胞划痕试验和Transwell检测细胞迁移和侵袭能力,用Western-blot检测凋亡相关因子Bax、p53、Bcl-2、Caspase-9、Caspase-3的表达。结果显示:相比MCF-7-HBLV(空载体组)和MCF-7细胞组(空白组),过表达组(MCF-7-ROP16Ⅱ)中ROP16 m RNA和蛋白表达显著升高;过表达组的细胞增殖率、侵袭率和迁移率显著降低(P<0.05);而细胞凋亡率显著升高(P<0.05);促凋亡因子Bax、p53、Caspase-3、Caspase-9高表达(P<0.05),而抗凋亡因子Bcl-2低表达(P<0.05)。结果表明,弓形虫Ⅱ型ROP16蛋白抑制MCF-7细胞增殖、迁移和侵袭,且诱导细胞凋亡。展开更多
基金Supported by the National Natural Science Foundation of China,No.81471983the Key Research and Development Plan Project of Anhui Province,Department of Science and Technology 2019,No.201904a07020043+1 种基金the Key Project of Natural Science Research in the Universities of Anhui Provence,No.KJ2017A202the Research Fund Project of Anhui Institute of Transforming Medicine,No.2017zhyx04
文摘BACKGROUND Inflammatory bowel disease（IBD） is characterized by chronic and non-specific inflammation of the intestinal mucosa and mainly includes ulcerative colitis and Crohn’s disease.AIM To explore the beneficial effect of Toxo ROP16Ⅰ/Ⅲ-induced M2 phynotype macrophages in homeostasis of IBDs through downregulation of M1 inflammatory cells.METHODS RAW264.7 macrophages stimulated by lipopolysaccharide（LPS）（M1 cells） were co-cultured with Caco-2 cells as an inflammatory model of IBD in vitro.The expression of Toxo ROP16Ⅰ/Ⅲ was observed in RAW264.7 macrophages that were transfected with p EGFP-rop16Ⅰ/Ⅲ.The phenotypes of M2 and M1 macrophage cells were assessed by quantitative real-time reverse transcriptase polymerase chain reaction and the expression of tumor necrosis factor（TNF）-α,interleukin（IL）-1β,IL-6,transforming growth factor（TGF）-β1,IL-10,inducible nitric oxide synthase（i NOS）,and arginase-1（Arg-1） was detected.The expression of i NOS,Arg-1,signal transducer and activator of transcription 3（Stat3）,p-Stat3,Stat6,pStat6,programmed death ligand-2（PD-L2）,caspase-3,-8,and-9 was analyzed by Western blotting,and Griess assays were performed to detect nitric oxide（NO）.TNF-α,IL-1β,IL-6,TGF-β1,and IL-10 expression in the supernatants was detected by enzyme-linked immunosorbent assay,and Caco-2 cell apoptosis was determined by flow cytometry after mixing M1 cells with M2 cells in a Caco-2 cell co-culture system.RESULTS M1 cells exhibited significantly increased production of i NOS,NO,TNF-α,IL-1β,and IL-6,while Toxo ROP16Ⅰ/Ⅲ induced macrophage bias to M2 cells in vitro,showing increased expression of Arg-1,IL-10 and TGF-β1 and elevated production of p-Stat3 and p-Stat6.The mixed M1 and M2 cell culture induced by Toxo ROP16 Ⅰ/Ⅲ exhibited decreased production of NO and i NOS and upregulated expression of Arg-1 and PD-L2.Accordingly,Caco-2 cells became apoptotic,and apoptosis-associated proteins such as caspase-3,-8 and-9 were dampened during co-cu
文摘为探讨弓形虫Ⅱ型(ME49)ROP16蛋白对人乳腺癌MCF-7细胞增殖、凋亡、迁移和侵袭的影响,利用Ⅱ型ROP16(MCF-7-ROP16Ⅱ)和空载体(MCF-7-HBLV)慢病毒载体分别感染MCF-7细胞,用实时荧光PCR、Western-blot验证ROP16蛋白在MCF-7细胞的表达,用CCK-8和流式细胞术检测细胞增殖和凋亡,用细胞划痕试验和Transwell检测细胞迁移和侵袭能力,用Western-blot检测凋亡相关因子Bax、p53、Bcl-2、Caspase-9、Caspase-3的表达。结果显示:相比MCF-7-HBLV(空载体组)和MCF-7细胞组(空白组),过表达组(MCF-7-ROP16Ⅱ)中ROP16 m RNA和蛋白表达显著升高;过表达组的细胞增殖率、侵袭率和迁移率显著降低(P<0.05);而细胞凋亡率显著升高(P<0.05);促凋亡因子Bax、p53、Caspase-3、Caspase-9高表达(P<0.05),而抗凋亡因子Bcl-2低表达(P<0.05)。结果表明,弓形虫Ⅱ型ROP16蛋白抑制MCF-7细胞增殖、迁移和侵袭,且诱导细胞凋亡。
