【摘要】 目的:通过体内外实验探讨锰超氧化物歧化酶(manganese superoxide dismutase,MnSOD)过量表达对食管癌TE-1细胞增殖的影响。方法:采用病毒感染法将不同剂量构建有MnSOD基因的重组质粒转入食管癌TE-1细胞,建立中、高表达MnSOD的TE-1细胞pLenti6-mMnSOD/TE-1(TE-1Mm)和pLenti6-hMnSOD/TE-1(TE-1Mh);采用RT-PCR法及Western印迹法检测转染MnSOD重组质粒的TE-1细胞中mRNA和蛋白水平的表达变化情况;应用平皿克隆形成实验及FCM法观察细胞增殖、凋亡和周期变化的情况;将MnSOD转染后的TE-1细胞接种到裸鼠皮下检测成瘤及肿瘤生长情况,采用免疫组织化学和Western印迹法检测移植瘤中MnSOD蛋白的表达水平。结果:建立稳定表达MnSOD蛋白的TE-1细胞株;RT-PCR及Western印迹法检测结果均证实,感染不同剂量的MnSOD重组质粒的TE-1细胞中,MnSOD的表达水平随感染剂量的增加而上升;细胞平皿克隆检测结果提示,TE-1Mm和TE-1Mh细胞的集落形成能力为(23.0±2.7)%和(45.3±4.5)%,分别低于和高于TE-1细胞的(34.7±4.2)%及TE-1n细胞的(33.7±4.7)%,实验组间比较及与对照组进行比较,差异均有统计学意义(P<0.05);AnnexinV-PI双染FCM检测结果显示,TE-1Mm和TE-1Mh细胞的早期细胞凋亡率为(10.6±1.0)%和(1.0±0.1)%,分别高于和低于对照组TE-1细胞的(2.6±0.2)%和TE-1n细胞的(2.5±0.3)%(P<0.05);细胞周期检测结果显示,MnSOD过量表达使G0/G1期细胞数在TE-1Mm细胞中增多,而在TE-1Mh细胞中减少,G2/M期和S期细胞数则在TE-1Mm细胞中减少,在TE-1Mh细胞中增多;与亲本TE-1细胞和感染空载体TE-1n细胞相比,TE-1Mm细胞处在增殖期细胞少,而TE-1Mh细胞处在增殖期细胞多(P<0.05)。TE-1Mm细胞接种裸鼠后,肿瘤生长慢,体积小,而接种TE-1Mh细胞则相反,肿瘤生长速度明显加快;瘤组织中的MnSOD蛋白的表达水平,与对照组比较差异均有统计学意义(P<0.05)。结论:MnSOD过量表达通过改变细胞周期和凋亡,对食管癌TE-1细胞的增殖和移植瘤的生长均表现为促进和抑制增殖的双向作用。更多还原

【Abstract】 Objective:To explore the effect of manganese superoxide dismutase(MnSOD) overexpression on the proliferation of esophageal cancer cell line TE-1 in vivo and in vitro.Methods:TE-1 cells were transfected with a plasmid binding MnSOD cDNA,and then TE-1Mm cells(with moderate expression of MnSOD) and TE-1Mh cells(with high expression of MnSOD) were established.RT-PCR and Western blotting were used to detect the mRNA and protein expressions of target MnSOD gene in both of TE-1Mm and TE-1Mh cells,respectively.The ability of cell proliferation and apoptosis and the cell cycle distribution were examined by plate colony-forming test and flow cytometry(FCM).The xenograft tumor models in nude mice were established.The effect of MnSOD overexpression on cell proliferation was evaluated in vivo,and the expression of MnSOD protein in xenograft tumors was detected by immunohistochemistry and Western blotting.Results:TE-1 cells with different expression levels of MnSOD proteins were established by transfection with different amounts of plasmids binding MnSOD cDNA.The colony-formation rates of TE-1Mm and TE-1Mh cells were(23.0±2.7)% and(45.3±4.5)%,respectively,which were both significantly higher than those in TE-1 cells(34.7±4.2)% and TE-1n cells(33.7±4.7)%,P<0.05.The apoptosis rate of TE-1Mm(10.6±1.0)% was significantly higher than those in TE-1 cells(34.7±4.2)% and TE-1n cells(33.7±4.7)%,and the apoptosis rate of TE-1Mh(10.6±1.0)% was significantly lower than those in TE-1 cells and TE-1n cells(P<0.05).FCM revealed that the percentage of TE-1Mh cells was decreased in G0/G1 phase and increased in G2/M and S phases,while the percentage of TE-1Mm cells was increased in G0/G1 phase and decreased in G2/M and S phases induced by MnSOD overexpression.The growth of xenograft tumors was inhibited in TE-1Mm cell-implanted group,while which was improved in TE-1Mh cell-implanted group.The expressions of MnSOD protein in TE-1Mm and TE-1Mh cells were significantly higher than those in TE-1 and TE-1n cells(P<0.05).Conclusion:MnSOD overexpression exerts a two-way effect involving inhibition or promotion on the proliferation of TE-1 cells in vivo and in vitro.更多还原