siRNA联合沉默MMP-9和FAK基因对小鼠黑色素瘤高转移细胞B16F10体外侵袭和迁移的影响  

Effect of siRNA Combined-silencing MMP-9 and FAK on Invasion and Migration of Mouse Melanoma Highly Metastatic Cells B16F10 in vitro

作　　者：汤禾静 唐照勇[1] 刘隆兴[1] 张小梅[2] 王祎婷[2] 方廖琼[1,2] 

机构地区：[1]西南大学生物技术学院,重庆400716 [2]重庆医科大学生物医学工程学院省部共建超声医学工程国家重点实验室超声医学工程重庆市市级重点实验室,重庆400016

出　　处：《中国生物工程杂志》2014年第9期40-47,共8页China Biotechnology

基　　金：国家“973计划”项目资助(2012CB722402)

摘　　要：目的:观察双基因联合干扰MMP-9和FAK对小鼠黑色素瘤高转移细胞B16F10体外侵袭、迁移能力的影响。方法:分别构建pGV102-MMP9-siRNA,pGV102-FAK-siRNA重组质粒载体,脂质体^(TM)2000介导转染小鼠黑色素瘤B16F10细胞,实验分为空白对照组、Anti-MMP-9组,AntiFAK组、Anti-MMP-9&FAK组、阴性对照组。经G418筛选GFP+克隆,流式细胞仪分析阳性率,激光共聚焦观察转染后细胞形态,半定量RT-PCR检测各组B16F10细胞MMP-9和FAK基因的mRNA转录水平,Transwell侵袭、迁移实验测定各组B16F10细胞体外侵袭、迁移能力。结果:经G418筛选,3个转染组阳性率分别为92.41±1.64%,95.72±0.21%,91.52±0.11%,且转染后细胞形态良好;与空白对照组相比,3个转染组的MMP-9,FAK mRNA转录水平下降明显(P<0.01),迁移、侵袭能力明显降低(P<0.01),但Anti-MMP-9&FAK组细胞侵袭迁移能力显著低于Anti-MMP-9组和Anti-FAK组(P<0.01)。结论:相比单独沉默MMP-9或FAK,联合沉默MMP-9和FAK可明显降低小鼠黑色素瘤B16F10细胞体外迁移、侵袭能力。Objective： To observe the effect of combined-silencing MMP-9 and FAK on invasion and migration of mouse melanoma highly metastatic B16F10 cells in vitro. Methods： Construct recombinant plasmid vectors pGV102-MMP9-siRNA and pGV102-FAK-siRNA respectively. Culture mouse melanoma B16F10 cells, then the plasmid vectors were transfected into cells using Lipofeetamine TM2000 method. This experiment was divided into 5 groups of blank control group, negative control group, Anti-MMP-9 experimental group, Anti-FAK experimental group, and Anti-MMP-9 ＆FAK experimental group. G418 was used to screen those GFP ＋ cells. In addition, the transfection efficiency were assayed with flow cytometry, and the cellular morphology of transfected cells were observed by confocal microscopy, mRNA level of MMP-9 and FAK of all groups B16F10 cells was detected by RT-PCR. Finally invasion and migration of transfected B16F10 ceils in vitro were tested by transwell experiment. Results Transfection efficiencies of the three experimental groups were to 92.41 ＋_ 1.64%, 95.72 ＋ 0.21%, 91. 52 -＋ 0. 11% respectively by flow cytometry via G418 screening. Moreover, screened cells morphology was good. Compared with the blank control group, MMP-9 and FAK mRNA level of three experimental groups＇ cells was significantly lowered （ P 〈 0.01 ） ; their invasion and migration also weakened dramatiely （P 〈0. 01 ）, and Anti-MMP-9 ＆FAK experimental group was very distinctly lower than Anti-MMP-9 experimental group and Anti-FAK experimental group. Conelusion Combined-silencing MMP-9 and FAK could obviously inhibit invasion and migration of mouse melanoma cell B16F10 in vitro.

关 键 词：MMP-9 FAK RNA 干扰 黑色素瘤 

分 类 号：Q343[生物学—遗传学]