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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。
OBJECTIVE: To investigate the effect of manganese superoxide dismutase (MnSOD) overexpression on the proliferation of esophageal cancer TE-1 cells in vitro and in vivo. Methods: Recombinant plasmids containing different MnSOD gene levels were transfected into esophageal cancer TE-1 cells by virus infection and pLenti6-mMnSOD / TE-1 (TE-1Mm) and pLenti6 The expression of mRNA and protein in TE-1 cells transfected with MnSOD recombinant plasmid was detected by RT-PCR and Western blotting. The results of plate clone formation and FCM The proliferation, apoptosis and cycle changes of cells were observed. TE-1 cells transfected with MnSOD were inoculated subcutaneously in nude mice to detect tumorigenesis and tumor growth. Immunohistochemistry and Western blotting were used to detect the expression of MnSOD protein The expression level. Results: The TE-1 cell line stably expressing MnSOD protein was established. The results of RT-PCR and Western blotting confirmed that the expression level of MnSOD in TE-1 cells infected with different doses of MnSOD recombinant plasmid increased with the increase of infection dose (23.0 ± 2.7)% and (45.3 ± 4.5)% of TE-1Mm and TE-1Mh cells were significantly lower than that of TE-1 cells (34.7 ± 4.2%) and TE-1n cells (33.7 ± 4.7%). The FCM results of Annexin V-PI double staining showed that the differences between the experimental groups and the control group were statistically significant (P < The rates of early apoptosis in -1Mm and TE-1Mh cells were (10.6 ± 1.0)% and (1.0 ± 0.1)%, respectively, higher than and lower than those in TE-1 cells (2.6 ± 0.2% (2.5 ± 0.3)% (P <0.05). The results of cell cycle assay showed that over-expression of MnSOD increased the number of G0 / G1 phase cells in TE-1Mm cells and decreased in TE- The number of M-phase and S-phase cells decreased in TE-1Mm cells and increased in TE-1Mh cells. Compared with parental TE-1 cells and empty vector TE-1n cells, TE-1Mm cells were in proliferating phase Less, whereas TE-1Mh cells proliferated Multi-cell (P <0.05). The growth of TE-1Mm cells was slow and the volume was small after inoculation of nude mice. On the contrary, the growth of TE-1Mh cells was accelerated obviously. The expression level of MnSOD protein in tumor tissue was statistically different from that of control group Significance (P <0.05). Conclusion: Overexpression of MnSOD can inhibit the proliferation of esophageal carcinoma TE-1 cells and the growth of xenografts by both cell cycle and apoptosis.