锰超氧化物歧化酶过量表达对食管癌细胞增殖的影响

Bidirectional effect of MnSOD overexpression on the proliferation of esophageal cancer cells in vitro

目的构建锰超氧化物歧化酶（MnSOD）基因重组慢病毒表达载体，并观察其对食管癌细胞增殖的影响。方法采用慢病毒转染法将MnSOD重组质粒转染食管癌TE-1细胞，建立MnSOD中、高表达的稳定转染细胞（TE-1Mm、TE-1Mh）及空载体细胞株（TE-1Mn细胞）。逆转录聚合酶链反应（RT．PCR）、免疫细胞化学法及Westernblot检测MnSOD在食管癌TE-1细胞中的表达；采用四甲基偶氮唑蓝（MTT）比色实验、平皿克隆形成实验、AnnexinV-FITC／PI双染实验及流式细胞术（FCM）研究MnSOD过表达在体外对TE．1细胞的影响。结果RT-PCR检测显示，转染后的TE-1细胞含有不同表达水平的MnSOD。免疫细胞化学及Westernblot显示，TE-1Mm细胞和TE-1Mh细胞含有不同表达水平的MnSOD蛋白。MTT法显示，与对照细胞（TE-1细胞、TE-1Mn细胞）相比，培养48h者TE-1Mm细胞的存活率下降，TE-1Mh细胞的存活率升高，组间比较差异具有统计学意义（P〈0．05）。TE一1Mm细胞平皿克隆集落形成能力为（23．0±2．7）％，TE-1Mh细胞为（45．3±4．5）％，而与TE一1细胞[（34．7±4．2）％]和TE．1Mn细胞[（33．7±4．7）％]相比，差异有统计学意义（P〈0．05）。TE-1Mm细胞早期凋亡率为（10．6±1．0）％，TE-1Mh细胞为（1．0±0．1）％，与TE-1细胞[（2．6±0．2）％]和TE-1Mn细胞[（2．5±0．6）％]相比，差异有统计学意义（P〈0．05）。TE-1Mm和TE-1Mh细胞线粒体凋亡荧光指数分别为0．948±0．019和1．076±0．022，与TE-1细胞（1．000±0．022）和TE-1Mn细胞（0．997±0．023）相比，差异有统计学意义（P〈0．05）。TE-1Mm细胞内活性氧荧光指数（0．859±0．040）低于TE-1细胞（1．000±0．042）和TE-1Mn细胞（1．002±0．047），但高于TE-1Mh细胞（0．763±0．039），差异均有统计学意义（P〈0．05）。结论MnSOD过表达对食管癌TE-1细胞增殖具有抑制和促进的双向作用。

Objective To construct a recombinant lentiviral vector for manganese superoxide dismutase （MnSOD） gene expression, and observe its effect on the proliferation of esophageal cancer cells in vitro. Methods Chemical methods were employed for synthesis of the MnSOD cDNA sequence sections, along with the attB sites. Target gene fragment was constructed on the pMD-18T vector, and the recombinant plasmid pDONR221 was obtained after BP recombination reaction. Sequencing was followed by LR recombination reaction between the plasmid and DEST to obtain the lentiviral vector, which worked with helper plasmid for co-transfection of human embryonic kidney epithelial cells （293T cells）. Amplification was done to determine its titer, and both transfection and selection procedures were made to get two stable transfected esophageal cancer TE-1 cell lines with medium MnSOD expression （ TE-1Mm cells） and high MnSOD expression （TE-1Mh cell ）, and empty vector cell （TE-1Mn cells ）. Reverse transcription polymerase chine reaction （RT-PCR）, immunofluorescence, immunocytochemistry and Western blot were used to detect the target gene with respect to its expression in the TE-1 cells. Additionally, colorimetric 3- [ 4,5-dimethy thiazol-2-yl ] -2,5-diphenyltetrazolium bromide （MTY） assay, agar colony formation assay, annexin V-FITC/PI staining and flow cytometry experiments were also conducted as to observe the influence of the medium and high MnSOD overexpressions on the proliferation of esophageal cancer cells. Results RT-PCR indicated that the transfected TE-1 cells showed positive MnSOD expression at different levels. , immunocytochemistry and Western blot suggested that TE-1Mm cells and TE-1Mh cells had MnSOD protein expression at different levels. MTT assay indicated that TE-1Mm cells had a significantly decreased survival rate compared with that of the two control cells （TE-1 cells and TE-1Mn cells）, and TE- 1 Mh cells had an significantly increased survival rate （ P 〈 0.05 ）. The colony formation ability of TE-1Mm cells was （23.0 ± 2.7 ）%, and that of TE-1Mh cells was （45.3 ± 4.5 ）%, significantly different form the （34.7±4.2）% in TE-1 ceils and （33.7 ±4.7）% in TE-1Mn cells （P〈0.05）. Annexin V-FITC/PI double staining experiment of the stably transfected cells cultured for 48 h showed that the early apoptosis rate in TE-1Mm cells was （ 10.6 ± 1.0 ） %, significantly higher than （ 2.6 ± 0.2 ） % in the TE-1 cells, （2.5±0.6）% in the empty vector cells and （1.0 ±0. 1）% in the TE-1Mh eels （P〈0.05）. The fluorescence index （FI） of mitoehondrial apoptosis of TE-1Mm cells was 0. 948 ±0.019, significantly lower than that of TE-1 cell （ 1. 000 ± 0.022 ） and empty vector The fluorescence index of TE-1Mn cells （0.997 ± 0.023 ） and TE-1 cells （ 1. 000 ± 0. 022） were significant different from that of 0.948 ± 0.019 in TE-1Mm cells and 1. 076 ± 0. 022 in TE-1Mh cells, indicating a significant difference of mitochondrial apoptosis between the cell groups. FCM results indicated that the ROS fluorescence index of TE-1Mm cells was 0. 859 ± 0.040, that of TE-1Mh cells was 0. 763 ±0. 039, significantly lower than that of TE-1 cells （1. 000 ±0.042） and empty vector cells （ 1. 002 ±0.047） （ P 〈 0.05 ）. Conclusions Stably transfected cell lines with MnSOD expression have been successfully established. MnSOD overexpression shows bidirectional effect on the proliferation of esophageal cancer cells.