玉米赤霉烯酮对趋化因子诱导的小鼠T细胞迁移效应和黏附与迁移相关蛋白表达的影响
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摘要
为揭示玉米赤霉烯酮(ZEA)免疫毒性机理,通过体外试验研究了ZEA对趋化因子诱导的小鼠T细胞迁移效应的影响,以及这一过程中与细胞黏附与迁移相关蛋白的变化。以Con A作为T细胞活化剂,以不同浓度ZEA(0、10、20、40μmol/L)染毒处理细胞后,Transwell法检测T细胞分别在CCL19、CCL21作用下的迁移效应,以及利用流式细胞术检测迁移后CD4、CD8阳性T细胞所占比例。激光共聚焦显微镜拍摄细胞穿膜形态,Western blot检测T细胞迁移及细胞黏附相关蛋白的表达。结果显示,ZEA可以分别降低CCL19和CCL21趋化的T细胞的迁移指数并扰乱迁移后CD4、CD8阳性T细胞之间的比例。此外,ZEA可以不同程度地抑制CCL19和CCL21介导的T细胞穿膜效应。Western blot结果显示,20、40μmol/L的ZEA作用可下调T细胞迁移及细胞黏附相关蛋白的表达。结果表明,ZEA可以抑制趋化因子介导的T细胞迁移效应,在这一过程中,黏附蛋白及迁移蛋白的表达均受到抑制,提示ZEA可能通过抑制T细胞运动过程中黏附及迁移这两个环节,进而影响T细胞的免疫应答,从而发挥免疫抑制作用。
In order to further reveal the mechanism of immunotoxicity of ZEA,the effect of ZEA on the cell migration of mouse T cells under the influence of chemokines and the changes of cell adhesion and migration protein in this process were studied by in vitro test.Con A as T cell activator and cells were treated with different concentrations of ZEA(0,10,20,40μmol/L),the migration effect of T cells under the action of CCL19,CCL21 was detected by Transwell assay and the percentages of CD4,CD8 positive T cells after migration were detected by flow cytometry.Laser confocal microscope was used to capture the morphology of cell penetrating membrane.Expressions of T cell migration and cell adhesion related proteins were detected by Western blot.The results showed that ZEA could decrease the migration index of T cells and disturb the ratio of CD4,CD8 positive T cells after migration,respectively.In addition,ZEA could inhibit the transmembrane effect of T cells mediated by CCL19,CCL21 to some extent.Western blot results showed that ZEA could down-regulate the expression of T cell migration and cell adhesion related proteins at 20,40μmol/L.The results showed that ZEA could inhibit the migration of T cells mediated by chemokine,and the activities of adhesion protein and migration protein were inhibited in this process.These results suggest that ZEA may play an immunosuppressive role by inhibiting the adhesion and migration of T cells in the process of movement,thus affecting the immune response of T cells.
In order to further reveal the mechanism of immunotoxicity of ZEA,the effect of ZEA on the cell migration of mouse T cells under the influence of chemokines and the changes of cell adhesion and migration protein in this process were studied by in vitro test.Con A as T cell activator and cells were treated with different concentrations of ZEA(0,10,20,40μmol/L),the migration effect of T cells under the action of CCL19,CCL21 was detected by Transwell assay and the percentages of CD4,CD8 positive T cells after migration were detected by flow cytometry.Laser confocal microscope was used to capture the morphology of cell penetrating membrane.Expressions of T cell migration and cell adhesion related proteins were detected by Western blot.The results showed that ZEA could decrease the migration index of T cells and disturb the ratio of CD4,CD8 positive T cells after migration,respectively.In addition,ZEA could inhibit the transmembrane effect of T cells mediated by CCL19,CCL21 to some extent.Western blot results showed that ZEA could down-regulate the expression of T cell migration and cell adhesion related proteins at 20,40μmol/L.The results showed that ZEA could inhibit the migration of T cells mediated by chemokine,and the activities of adhesion protein and migration protein were inhibited in this process.These results suggest that ZEA may play an immunosuppressive role by inhibiting the adhesion and migration of T cells in the process of movement,thus affecting the immune response of T cells.
引文
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[4] Zhang K,Tan X,Li Y,et al.Transcriptional profiling analysis of Zearalenone-induced inhibition proliferation on mouse thymic epithelial cell line 1[J].Ecotoxicol Environ Safety,2018,153:135-141.
[5] Sharfe N,Freywald A,Toro A,et al.Ephrin stimulation modulates T cell chemotaxis[J].Eur J Immunol,2002,32(12):3745-3755.
[6] Baggiolini M,Dewald B,Moser B.Human chemokines:An update[J].Annual Rev Immunol,1997,15(1):675-705.
[7] Wiedle G,Dunon D,Imhof B A.Current concepts in lymphocyte homing and recirculation[J].Critical Rev Clin Lab Sci,2001,38(1):1-31.
[8] Newman W.Chemokines and their receptors.[J].Drug Discovery Today,1999,4(7):299-300.
[9] Mangeat P,Roy C,Martin M.ERM proteins in cell adhesion and membrane dynamics[J].Trends Cell Biol,1999,9(5):187-192.
[10] Pertz O,Hodgson L,Klemke R L,et al.Spatiotemporal dynamics of RhoA activity in migrating cells[J].Nature,2006,440(7087):1069-1072.
[11] Humphries J D,Wang P,Streuli C,et al.Vinculin controls focal adhesion formation by direct interactions with talin and actin[J].J Cell Biol,2007,179(5):1043-1057.
[12] 蔡国栋,孙凯,项自来,等.玉米赤霉烯酮对小鼠T淋巴细胞体外活化、增殖的影响[J].畜牧兽医学报,2017,48(7):1357-1364.
[13] Kim H,Williams N I,Kazlauskas A,et al.Regulation of chemotaxis by the platelet-derived growth factor receptor-beta[J].Science,1989,243(4898):474-476.
[14] Mackay C R.Chemokine receptors and T cell chemotaxis.[J].J Exper Med,1996,184(3):799-802.
[15] Springer T A.Traffic signals for lymphocyte recirculation and leukocyte emigration:The multistep paradigm[J].Cell,1994,76(2):301-314.
[16] Wurtzel J G T,Kumar P,Goldfinger L E.Palmitoylation regulates vesicular trafficking of R-Ras to membrane ruffles and effects on ruffling and cell spreading[J].Small GTPases,2012,3(3):139-153.
[17] Baekkevold E S,Yamanaka T,Palframan R T,et al.The Ccr7 ligand ELC (Ccl19) is transcytosed in high endothelial venules and mediates T cell recruitment[J].J Exper Med,2001,193(9):1105-1112.
[18] Mueller S N,Germain R N.Stromal cell contributions to the homeostasis and functionality of the immune system[J].Nat Rev Immunol,2009,9(9):618-629.
[19] Wang X J,Yan W M,Lu Y L,et al.CD4-CD8-T cells contribute to the persistence of viral hepatitis by striking a delicate balance in immune modulation[J].Cell Immunol,2012,280(1):76-84.