青藤碱对人胰腺癌细胞系PANC-1侵袭转移的影响

摘要

目的:体外实验探讨青藤碱对胰腺癌细胞PANC-1侵袭和迁移能力的影响.方法:用高糖培养基DMEM培养胰腺癌PANC-1细胞,取对数期生长的细胞,实验组用0.5、1.0 mmol/L青藤碱处理,运用侵袭和迁移实验研究青藤碱对胰腺癌侵袭转移的影响,采用激光共聚焦实验观察青藤碱对细胞骨架的改变,免疫印迹技术检测青藤碱对胰腺癌细胞PANC-1 E-钙粘着蛋白Nimentin和波形蛋白(E-cadherin)表达水平的改变.结果:实验组PANC-1细胞穿过铺有Matrigel胶小室的细胞数量显著减少,差异有统计学意义(P＜0.05);细胞划痕实验表明,青藤碱处理24 h和48 h的胰腺癌细胞迁移速度明显小于对照组(P＜0.05);激光共聚焦显示,实验组PANC-1细胞内绿色荧光染色的F-actin与对照组比较显著减少,且荧光强度也较弱(P＜0.05);β-微管蛋白(β-tubulin)红色荧光强度明显减弱,差异有统计学意义(P＜0.05);免疫印迹结果显示,实验组Vimentin逐渐减少,E-cadherin逐渐增多,与对照组比较差异具有统计学意义(P＜0.05).结论:一定浓度的青藤碱可抑制体外胰腺癌PANC-1细胞的侵袭和转移能力.%Objective:To study the effect of sinomenine on invasion and migration of pancreatic cancer cell PANC-1.Methods:PANC-1 cells were cultured in vitro,and treatedwith different concentrations of sinomenine (0,0.5,1.0 mmol/L) at the growing period of cells,Transwell method and wound healing experiment were adopted to test the invasion and migration of Panc-1 cells;The influence of SIN on cytoskeleton was detected with immunofluorescence technique;The changes of EMT-associated protein levels of Panc-1 cells after exposed with different concentrations of SIN were detected by Western Blotting.Result:Transwell experiment indicated the passing Panc-1 cell numbers in experiment groups were obviously less than that in control group (P ＜ 0.05).Wound healing experiment discovered that in 24h and 48h after infection,Panc-1 cells in control group migrated obviously faster than those in experiment groups.Immunofluorescence test showed that in SIN untreated Panc-1 cells,the parallel arrangement and cross overlapping reticulated bundle fibers formed by F-actin microfilament appeared in the whole cell and a lot of microfilaments penetrated deeply in cell processes.In comparison with control group,the F-actin of experiment group which stained with FITC-phalloidin was obviously decreased,and the microfilaments in kytoplasm became shorter or disappeared or became rarefied and arranged irregularly.Compared with the control group,the length of the microtubules were much shorter,bundle structure disapperaed or ruptured and the red fluorescence intensity was significantly reduced.Westernblot indicated that the expression of vimentin was significantly decreased and E-cadherin greatly enhanced compared with the control group.Conclusion:These results suggest that sinomenine could inhibit the invision and migration of human pancreatic cancer cell line PANC-1.