小RNA干扰Akt2对U87恶性胶质瘤细胞迁移和侵袭能力的影响  

In vitro study of Akt2 function on U87 malignant glioma cells migration and invasion

作　　者：张宝刚[1,2] 谷峰[3] 李文良[4] 郭华[1] 张宁[1] 马勇杰[1] 

机构地区：[1]天津医科大学附属肿瘤医院研究所中心实验室,天津300060 [2]潍坊医学院乳腺病理研究室,潍坊261053 [3]天津医科大学附属肿瘤医院乳腺病理研究室,天津300060 [4]天津医科大学附属肿瘤医院颅脑肿瘤科,天津300060

出　　处：《解剖学报》2009年第5期691-695,共5页Acta Anatomica Sinica

基　　金：国家重点基础研究发展计划973项目子课题资助项目(2006CB705600);国家自然科学基金资助项目(30700253和30800355);长江学者和创新团队发展计划资助项目(IRT0743);教育部留学归国人员科研启动基金资助项目;天津医科大学基金项目资助(2006KY21);天津医科大学附属肿瘤医院引进人才启动基金资助项目

摘　　要：目的探讨转染Akt2-siRNA表达载体阻断Akt2信号转导通路对U87恶性胶质瘤细胞运动、迁移和侵袭能力的影响。方法利用siRNA技术,稳定转染恶性胶质瘤细胞系U87;用RT-PCR和免疫印迹技术分别检测Akt2的mRNA和蛋白的表达;划痕实验和体外侵袭实验分别检测恶性胶质瘤细胞的运动和侵袭能力;用细胞黏附实验检测细胞的黏附能力,纤维型肌动蛋白(F-actin)聚合实验检测F-actin的聚合能力;免疫印迹技术检测LIMK的磷酸化,证明Akt2影响F-actin聚合的机制。结果稳定转染siAkt2载体后,Akt2的mRNA和蛋白水平均下降;细胞向划痕移动的速度比对照组慢(P<0.05);侵袭并穿透基质胶(Matrigel)的细胞数量比对照组少(P<0.01);细胞内F-actin聚合比对照组减少(P<0.05);黏附实验结果显示,黏附5min、15min后,低表达Akt2的细胞比对照组的黏附数量均减少(P<0.05)。结论Akt2基因沉默可以通过降低F-actin聚合、降低黏附于细胞外基质的能力而降低恶性胶质瘤细胞的运动和侵袭能力。Objective To detect the effect of Akt2 on U87 glioblastoma cells migration and invasion by Akt2 small RNA interference technology in vitro. Methods Akt2 expression was knocked down in U87 glioblastoma cells by Akt2 siRNA, stable clones were used to perform wound healing assay, F-actin polymerization assay, adhesion assay and matrigel invasion assay. Western blotting was used to check the expression level of phosphorylated cofilin and LIMK. Results Result of wound healing assay suggested that motility of U87 cells was significantly decreased with deficiency of Akt2 ; U87 cells with Akt2 deficiency was not capable of assembling actin effectively compared to control cells by F-actin polymerization assay. Further, phosphorylated cofilin and LIMK expression which are important for cellular motility were all decreased with Akt2 deficiency. Besides, adhesion and invasion ability of U87 cells were significantly impaired after Akt2 was knocked down. Conclusion Akt2 is important for U87 cells migration and invasion. Akt2 deficiency will lead to impairment of U87 glioblastoma cells motility, adhesive and invasive ability in vitro.

关 键 词：胶质瘤 细胞运动 侵袭 小RNA干扰 免疫印迹法 

分 类 号：R739.41[医药卫生—肿瘤]