MT2-MMP基因沉默对缺氧条件下培养的胰腺癌AsPC-1细胞增殖、凋亡及侵袭力的影响  

Effects of MT2-MMP silencing on the proliferation, apoptosis and invasion of pancreatic cancer cell AsPC-1 under hypoxia culture

作　　者：朱世凯[1] 周玉[1] 范平[2] 王博[2] 吴汉青[2] 杨洪吉[1] 熊炯炘[2] 吴河水[2] 

机构地区：[1]四川省医学科学院四川省人民医院器官移植中心肝胆胰外科,成都610072 [2]华中科技大学同济医学院附属协和医院胰腺疾病研究所

出　　处：《中华胰腺病杂志》2013年第6期378-381,共4页Chinese Journal of Pancreatology

摘　　要：目的 利用特异性siRNA沉默人胰腺癌AsPC-1细胞的膜型金属蛋白酶2（MT2-MMP）基因表达,观察其对缺氧条件下培养的细胞增殖、凋亡和侵袭力的影响.方法 采用脂质体转染法将插入MT2-MMP特异性siRNA的真核表达质粒转染至人胰腺癌AsPC-1细胞株（siRNA组）,以转染过表达MT2-MMP质粒（过表达组）或阴性对照质粒（空载体组）的AsPC-1作为对照.实时定量PCR法和蛋白质印迹法检测转染细胞的MT2-MMP mRNA和蛋白的表达.在缺氧条件（37℃、1％O2、5％ CO2、饱和湿度的三气培养箱）下培养后,应用CCK-8法检测转染细胞的增殖,流式细胞仪检测细胞的凋亡,Transwells小室检测细胞的侵袭能力.结果 成功获取MT2-MMP基因沉默的AsPC-1细胞株和过表达的细胞株.缺氧条件下培养24h后,空载体组、过表达组、siRNA组细胞增殖的吸光度值（A490值）分别为0.68±0.08、0.80 ±0.08、0.52±0.07;细胞凋亡率分别为（6.2±1.5）％、（2.8±1.1）％、（21.4±3.9）％;每个视野（200倍）的穿膜细胞数分别为（115.8 ±23.2）、（256.4±38.6）、（45.8±18.2）个.siRNA组较空载体组的细胞增殖显著被抑制,穿膜细胞数显著减少,而细胞凋亡率显著增加（P值均＜0.05）.过表达组较空载体组的细胞增殖显著增强,穿膜细胞数显著增加,而细胞凋亡率显著降低（P值均＜0.05）.结论 MT2-MMP基因沉默的AsPC-1细胞在缺氧条件下培养后细胞凋亡增加,增殖被抑制,侵袭力减弱.Objective To investigate the effects of MT2-MMP silencing by specific siRNA on pancreatic cancer cell AsPC-1 proliferation,apoptosis and invasion under hypoxia culture.Methods MT2-MMP siRNA plasmids were stably transfected into AsPC-1 cells （siRNA group） by liposome transfection method,AsPC-1 cells tranfected with and over-expressed MT2-MMP siRNA plasmids （over-expression group） and negative control plasmid （empty vector group） were treated as control.Real-time RT-PCR and Western Blot assay were used to detect the expression of MT2-MMP mRNA and protein.Under culture with hypoxia （37℃,1% O2,5% CO2,three gas saturated humidity incubator）,CCK-8,flow cytometry （FCM） and Transwell assays were applied to measure pancreatic cancer cell proliferation,apoptosis,and invasion.Results MT2-MMP silencing AsPC-1 and over-expressed AsPC-1 were successfully established.After culture with hypoxia for 24 h,the absorbance values （A490） in empty vector group,over-expression group and siRNA group were 0.68 ± 0.08,0.80 ± 0.08,0.52 ± 0.07 ; the apoptotic rates were （6.2 ± 1.5） %,（2.8 ± 1.1） %,（21.4±3.9）%,and the numbers of invasion cells in each field （200 times） were （115.8±23.2）,（256.4 ± 38.6）,（45.8 ± 18.2）.The proliferation in MT2-MMP siRNA group was significantly inhibited,the numbers of invasion cells was significantly decreased,but the apoptotic rates were obviously increased when compared with those in empty vector group （P 〈0.05）.The proliferation in over-expression group was significantly enhanced,the numbers of invasion cells was significantly increased,but the apoptotic rates were obviously decreased when compared with those in empty vector group （P 〈 0.05）.Conclusions AsPC-1 with MT2-MMP silencing could inhibit pancreatic cancer cells proliferation,induce cells apoptosis and attenuate tumor invasion under hypoxia culture.

关 键 词：胰腺肿瘤 基质金属蛋白酶类 RNA 小分子干扰 缺氧 细胞凋亡 肿瘤浸润 

分 类 号：R735.9[医药卫生—肿瘤]