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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2014, Vol. 34 Issue (9): 40-47    DOI: 10.13523/j.cb.20140907
研究报告     
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
汤禾静1, 唐照勇1, 刘隆兴1, 张小梅2, 王祎婷2, 方廖琼1,2
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
Effect of siRNA Combined-silencing MMP-9 and FAK on Invasion and Migration of Mouse Melanoma Highly Metastatic Cells B16F10 in vitro
TANG He-jing1, TANG Zhao-yong1, LIU Long-xing1, ZHANG Xiao-mei2, WANG Yi-ting2, FANG Liao-qiong1,2
1. College of Biotechnology, Southwest University, Chongqing 400016, China;
2. College of Biomedical Engineering, Chongqing Medical University State Key Laboratory of Ultrasound Engineering in Medicine Co-founded by Chongqing and the Ministry of Science, Chongqing 400016, China
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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TM2000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，Anti-FAK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。
关键词： MMP-9;  FAK;  RNA干扰;  黑色素瘤    
Abstract:

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 Lipofectamine 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 cells 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 dramaticly (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.
Key words: MMP-9    FAK    RNA interference    Malignant melanoma
收稿日期: 2014-06-25 出版日期: 2014-09-25
ZTFLH:  Q343  
基金资助:

国家“973计划”项目资助（2012CB722402）
通讯作者: 方廖琼     E-mail: lqfang06@163.com
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引用本文:

汤禾静, 唐照勇, 刘隆兴, 张小梅, 王祎婷, 方廖琼. 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[J]. 中国生物工程杂志, 2014, 34(9): 40-47.

TANG He-jing, TANG Zhao-yong, LIU Long-xing, ZHANG Xiao-mei, WANG Yi-ting, FANG Liao-qiong. Effect of siRNA Combined-silencing MMP-9 and FAK on Invasion and Migration of Mouse Melanoma Highly Metastatic Cells B16F10 in vitro. China Biotechnology, 2014, 34(9): 40-47.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20140907    或    https://manu60.magtech.com.cn/biotech/CN/Y2014/V34/I9/40


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