摘要目的 观察分别及联合抑制Rac及Rho通路后3D水凝胶中恶性胶质瘤细胞的侵袭迁移运动形式与整合素表达的变化. 方法 应用脂质体介导pCMVLifeAct-TagGFP2质粒转染U87细胞,将转染后细胞分为NSC23766处理组、Y-27632处理组、NSC23766和Y-27632联合处理组、空白对照组,前3组分别加入100 nmol/mL NSC23766、10 nmol/mLY-27632、100 nmol/mLNSC23766+10 nmol/mL Y-27632,空白对照组加入等量培养基.将4组细胞在水凝胶中进行3D培养,共聚焦显微镜观察4组中类圆形细胞、梭形细胞的组成及间质运动和变形虫运动形式的转换;将接种了4组细胞的水凝胶注入细胞趋化载玻片中,于活细胞工作站观察4组细胞的侵袭迁移运动形式并计算运动速度、运动距离;免疫荧光染色观察4组细胞整合素oV33的表达. 结果 3D水凝胶培养的U87细胞具有类梭形、类圆形2种形态,相应地表现为间质运动和变形虫样运动形式.与空白对照组比较,NSC23766处理组中类圆形细胞所占比例较高,Y-27632处理组中梭形细胞所占比例较高,差异均有统计学意义(P＜0.05).NSC23766处理组细胞的间质-变形虫运动(MAT)转换率为50.0％,Y-27632处理组变形虫-间质运动(AMT)转换率为42.8％.趋化实验结果显示NSC23766处理组、空白对照组、Y-27632处理组、NSC23766和Y-27632联合处理组细胞的运动速度和运动距离依次降低,差异有统计学意义(P＜0.05).免疫荧光染色检测显示Y-27632处理组细胞整合素αVβ3表达显著高于其他3组,空白对照组细胞整合素αV33表达显著高于NSC23766处理组和联合处理组. 结论 3D水凝胶可以作为U87细胞培养及三维形态观察的良好基质,Rac1与RohA通路靶向抑制剂的联合应用可作为控制脑胶质瘤播散的策略.

abstractsObjective To observe the changes of invasion and migration patterns and integrin expression of malignant glioma cells when Rac and Rho pathways are suppressed,respectively,in 3D hydrogel.Methods Liposome mediated pCMVLifeAct-TagGFP2 plasmids were transfected into glioma U87 cells,and then,these cells were divided into NSC23766 treatment group,Y-27632 treatment group,NSC23766 and Y-27632 combined treatment group,and control group.The three treatment groups were added NSC23766 (100 nmol/mL),Y-27632 (10 nmol/mL),100 nmol/mL NSC23766 and 10 nmol/mL Y-27632,respectively;the cells in the control group were added the same amount of medium.Cells were cultured in hydrogel;the composition of round cells,spindle cells and the mesenchymal and amoeboid cell movement transition were observed under confocal microscopy.The hydrogels of each group were infused into the microslide,and the cell chemotaxis effect were recorded and the cell movement velocities and distances were calculated in the living cell workstation.Immunofluorescence was used to observe the alternation ofintegrin expression.Results U87 cells cultured in 3D hydrogel exhibited spindle-like and round-like shapes,corresponding to mesenchymal and amoeboid cell movement.As compared with that in the control group,the proportion of round cells in the NSC23766 treatment group was significantly higher,and that of spindle cells in the Y-27632 treatment group was significantly higher (P＜0.05).The conversion rate ofmesenchymal-amoeboid transition was 50.0％ in NSC23766 treatment group and amoeboid mesenchymal transition was 42.8％ in Y-27632 treatment group as compared with that in the control group,with significant differences (P＜0.05).The velocity and distance of cells cultured in 3D hydrogel decreased orderly in NSC23766 treatment group,control group,Y-27632 treatment group and combined treatment group in chemotaxis test.The immunofluorescence test showed that integrin expression in the Y-27632 treatment group was significantly higher than that in the other three groups,and that in the control group was statistically higher than that in the NSC23766 treatment group and combined treatment group (P＜0.05).Conclusions 3D hydrogel can be used as a favorable substrate for cell culture.The combination targeted inhabitation of Rac 1 and RohA pathways provides theoretical basis for anti-invasion treatment against glioma.