MnSOD过量表达与BSO对食管癌细胞的增敏效应

基础医学与临床 ›› 2011, Vol. 31 ›› Issue (12): 1314-1319.

MnSOD过量表达与BSO对食管癌细胞的增敏效应

孙国贵

河北省唐山市人民医院

收稿日期:2011-07-28 修回日期:2011-10-28 出版日期:2011-12-05 发布日期:2011-12-05
通讯作者: 孙国贵 E-mail:sgg1980@tom.com
基金资助:
国家自然科学基金（30540005）

Radiosensitizative effect of MnSOD and BSO on esophageal cell

Received:2011-07-28 Revised:2011-10-28 Online:2011-12-05 Published:2011-12-05

摘要/Abstract

摘要： [摘要] 目的研究过量表达锰超氧化物歧化酶（manganese superoxide dismutase, MnSOD）在丁胱磺酰亚胺（BSO）作用下对食管癌TE-1细胞放射敏感性的影响。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，建立过量表达MnSOD的稳定转染细胞（TE-1Mm）及空载体细胞（TE-1Mn）。逆转录聚合酶链反应（reverse transcription polymerase chine reaction，RT-PCR）、Western blot检测转染后TE-1细胞中MnSOD mRNA和蛋白的表达。平皿克隆形成实验观察MnSOD过量表达对细胞增殖的影响。四甲基偶氮唑蓝（MTT）比色实验评价BSO、放射线及二者联合对食管癌细胞放射增敏的影响。流式细胞术（FCM）及Western blot检测细胞凋亡、活性氧（ROS）荧光强度及蛋白表达的变化。结果建立稳定过量表达MnSOD的TE-1细胞株；RT-PCR及Western blot证实转染MnSOD的TE-1细胞中含有过量表达水平的目的基因。TE-1Mm细胞平皿克隆集落形成能力23.0±2.7%，低于TE-1细胞34.7±4.2%；组间相比差异具有统计学意义（P＜0.05）。FCM实验显示：TE-1Mm细胞的早期细胞凋亡率10.6±1.0%，高于TE-1（2.6±0.2%）；组间相比差异具有统计学意义（P＜0.05）。 0.1～2.5 mg/L BSO及1、2、4、6、8Gy放射线处理48 h，TE-1Mm细胞生长抑制率明显增加，细胞生长明显减慢。经多靶单击拟合细胞存活曲线，TE-1Mm细胞的D0、Dq及N值相对减小，SER值增加。FCM显示，TE-1Mm细胞ROS荧光指数分别为0.81±0.04，与母细胞TE-1（1.00±0.06）相比，差异具有统计意义（P＜0.05）。Western blot结果显示，在相同时间（48 h）作用下，TE-1Mm细胞MnSOD蛋白表达在处理组相对量为0.19±0.02，与TE-1细胞相应蛋白表达量（0.11±0.01）相比差异具有统计学意义（P＜0.05）。结论MnSOD过量表达与BSO通过抑制增殖、诱导凋亡、下调细胞内ROS浓度提高食管癌细胞放射敏感性。

关键词: 锰超氧化物歧化酶, 丁胱亚磺酰亚胺, 食管肿瘤, 放射增敏

Abstract: 【Abstract】Objective To explore effect of manganese superoxide dismutase (MnSOD) overexpression on rediosensitivity in the esophageal cancer cell line in action of buthionine sulfoximine (BSO). Methods Overexpression of MnSOD cDNA by lentivirus to get stable transfected TE-1 cell lines of MnSOD overexpression (TE-1Mm). Reverse transcription polymerase chine reaction (RT-PCR）and Western blot were then introduced to detect the target gene with respect to its expression in esophageal TE-1 cells. Plating efficiency was also conducted as to the influence of MnSOD overexpression that might be found on TE-1 cells proliferation. Colorimetric 3-[4,5-dimethy thiazol-2-yl]-2, 5-diphenyltetrazolium bromide(MTT) assay, flow cytometry and western blot were also conducted as to the influence of the MnSOD overexpression that might be found on proliferation of TE-1 cells treated with BSO and radiation. Results: RT-PCR and Western blot showed that TE-1 cells transfected with MnSOD contained the target genes in overexpression. Plating efficiency was reduced in the MnSOD overexpression transfected cell line and decreased the plating efficiency to 23.0±2.7%. While, the TE-1 cell had a higher plating efficiency (34.7±4.2%). Furthermore, apparent apoptosis of TE-1Mm cell induced by transfected moderate MnSOD cDNA (10.6±1.0%) was indicated by Annexin V-FITC/PI staining compared to TE-1 cell (2.6±0.2%). Moreover, in multitarget single-hit model-fitting survival curves, the values of D0, Dq, N, and SER in TE-1Mm cell lines were less than in TE-1 cell. FCM results indicated that TE-1Mm of reactive oxygen species (ROS) fluorescence index were 0.81±0.04 compared to TE-1 cell (1.00±0.06). Western blot demonstrated that TE-1Mm had a higher MnSOD protein expression than those of TE-1 treated with BSO and radiation. Conclusion MnSOD overexpression could increase the radiosensitivity of esophageal cancer cell by inhibiting cell proliferation, inducing cell apoptosis and decreasing ROS concention in combination with BSO.

Key words: Manganese superoxide dismutase, Buthionine sulfoximine, esophageal carcinoma, radiosensitivity

孙国贵. MnSOD过量表达与BSO对食管癌细胞的增敏效应[J]. 基础医学与临床, 2011, 31(12): 1314-1319.