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肿瘤的发生是多基因、多因素共同作用的复杂生物学过程,迄今为止还不清楚各种肿瘤的明确病因。研究表明,DNA损伤修复的能力在一定程度上决定了肿瘤的发生[1-2]。及时、有效的DNA修复,有利于维持生物体基因组的完整性和抑制肿瘤的发生。人类X射线修复交叉互补基因(X-ray repair cross complementary gene,XRCC)家族最初因其具有对哺乳动物细胞电离辐射损伤的防护作用而被发现,是辐射损伤后DNA修复不可缺少的基因[3]。XRCC2是XRCC家族成员之一,通过DNA同源重组修复途径参与DNA双链断裂重接。本研究采用XRCC2基因沉默结肠癌细胞系,观察XRCC2基因沉默对结肠癌细胞体外增殖能力的影响,阐明XRCC2基因沉默与结肠癌细胞辐射敏感性的关系。
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如图 1所示,稳定转染的shRNA-XRCC2细胞中XRCC2蛋白表达明显下降,与对照组细胞和shRNA-SC组细胞相比,约分别减少了70%和65%。结果表明,XRCC2 shRNA质粒转染T84细胞后,有效地抑制了XRCC2蛋白的表达,成功建立了稳定的XRCC2基因沉默的结肠癌细胞系(T84/ XRCC2-shRNA)。
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对照组和shRNA-SC组细胞稳定生长,生长的速度基本一致;而shRNA-XRCC2组细胞于培养的第3天开始,与对照组和shRNA-SC组细胞相比,增殖速度明显减慢。不同培养时间结肠癌T84细胞的生长抑制率显示,从培养的第3天开始,shRNA-XRCC2组细胞生长抑制率一直平稳地维持在50%左右,明显低于shRNA-SC组(t=17.62、12.84、9.24,P < 0.05),见表 1。结果表明,沉默XRCC2表达有效地抑制了体外结肠癌T84细胞的增殖。
培养时间(d) 抑制率 shRNA-SC shRNA-XRCC2 1 1.1±0.2 5.3±1.6 3 5.3±1.5 46.7±8.0 5 14.8±3.5 49.9±8.8 7 22.1±4.1 49.7±9.5 注:表中,XRCC2:X射线修复交叉互补基因2。 表 1 不同培养时间结肠癌T84细胞的生长抑制率(%,x±s)
Table 1. The growth inhibition rate at different culture time of colorectal cancer T84 cells(%, x±s)
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从图 2可见,对照组和shRNA-SC组细胞克隆形成数目基本一致,分别为498.5±43.5和512.3±38.5;shRNA-XRCC2组和8 Gy组的细胞克隆形成数目明显减少,分别为422.7±43.4和389.5±24.4,与对照组细胞相比,差异有统计学意义(t=3.02、5.35,P均 < 0.05);shRNA-XRCC2+8 Gy组细胞克隆形成数最少(223.3±32.9),与对照组细胞克隆形成数相比,两者之间的差异有统计学意义(t=12.34,P < 0.01),与shRNA-XRCC2组和8 Gy组相比,差异有统计学意义(t=8.96、9.92,P均 < 0.01)。结果表明,XRCC2基因沉默联合辐射降低了结肠癌T84细胞的克隆形成能力。
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从流式分析结果可见,8 Gy X射线照射后,shRNA-XRCC2组停留在G2/M期的细胞增加(38.51±4.15)%,与对照组和shRNA-SC组相比,差异有统计学意义(t=3.92、5.49,P均 < 0.05),而G0/G1期和S期细胞减少;shRNA-XRCC2组细胞的凋亡率最高,达到(33.16±2.69)%,约是对照组和shRNA-SC组的5倍(t=15.31、12.78,P均 < 0.01)(表 2)。结果表明,XRCC2基因沉默联合辐射促使结肠癌T84细胞主要阻滞在G2/M期,大量细胞发生细胞凋亡。
组别 细胞周期 细胞凋亡率 G0/G1 G2/M S 对照组 48.83±1.61 26.20±3.50 24.97±4.82 6.90±1.40 shRNA-SC组 67.58±1.16 24.64±1.38 7.78±0.42 7.76±2.15 shRXNRAC-C2组 41.13±7.85 38.51±4.15 20.36±11.47 33.16±2.69 注:表中,XRCC2:X射线修复交叉互补基因2。 表 2 XRCC2基因沉默联合辐射后T84细胞的细胞周期和细胞凋亡率(%,x±s)
Table 2. The cell cycle distribution and cell apoptosis of T84 cells of silencing XRCC2 gene combined with radiation(%, x±s)
沉默XRCC2基因表达联合电离辐射对结肠癌细胞增殖能力的影响
The effect of silencing XRCC2 gene combined with ionizing radiation on growth of colorectal cancer cells
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摘要: 目的阐明shRNA干扰沉默X射线修复交叉互补基因2(XRCC2)对体外结肠癌T84细胞辐射敏感性的影响。方法采用MTT法检测稳定表达XRCC2基因沉默的结肠癌T84细胞的生长并计算细胞生长抑制率,克隆形成实验检测经X射线照射后T84细胞的克隆形成能力,采用流式细胞术检测经X射线照射后T84细胞的细胞周期和细胞凋亡率。结果shRNA-XRCC2组细胞于培养的第3天开始,增殖速度明显减慢,细胞生长抑制率平稳地维持在50%左右,明显低于shRNA-SC组(t=17.62、12.84、9.24,P < 0.05)。shRNA-XRCC2组和8 Gy组的细胞克隆形成数目分别为422.7±43.4和389.5±24.4,shRNA-XRCC2+8 Gy组细胞克隆形成数最少(223.3±32.9),与shRNA-XRCC2组和8 Gy组相比,差异有统计学意义(t=8.96、9.92,P < 0.01)。shRNA-XRCC2组停留在G2/M期的细胞增加(38.51±4.15)%,与对照组相比,差异有统计学意义(t=3.92,P < 0.05);shRNA-XRCC2组细胞的凋亡率最高,达(33.16±2.69)%,与对照组相比,差异有统计学意义(t=15.31,P < 0.01)。结论XRCC2基因沉默有效地抑制了体外结肠癌T84细胞的增殖,XRCC2基因沉默联合辐射促使T84细胞阻滞在G2/M期并发生细胞凋亡,从而提高了T84细胞对辐射的敏感性。
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关键词:
- 辐射 /
- 结肠肿瘤 /
- RNA干扰 /
- X射线修复交叉互补基因2
Abstract: Objective To investigate the effect of silencing X-ray repair cross complementary gene 2(XRCC2) through shRNA interference on the radiosensitivity of colorectal cancer cells. Methods The growth of colorectal cancer T84 cells of silencing XRCC2 was determined with MTT assay and the growth inhibition rate of T84 cells was detected. The ability to form colonies of T84 cells after exposure to X-ray radiation was examined with colony formation assay. The cell cycle distribution or cell apoptosis of T84 cells irradiated with X-ray radiation was performed by flow cytometric analysis. Results The cell growth of shRNA-XRCC2 group slowed down markedly from the third day of cell culture and the growth inhibition rate of shRNA-XRCC2 group steadily maintained about 50%, which was significantly less than that of shRNA-SC group(t=17.62, 12.84, 9.24, all P < 0.05). The number of colonies formed in shRNA-XRCC2 cells and 8 Gy radiation cells was 422.7±43.4 and 389.5±24.4 respectively. The number of colonies in shRNA-XRCC2+8 Gy radiation group was the least(223.3±32.9), which was decreased significantly compared with that of shRNA-XRCC2 group or 8 Gy group(t=8.96 and 9.92, both P < 0.01). Cells of shRNA-XRCC2 group arrested at G2/M phase were increased, reached(38.51±4.15)%, significantly higher than that of control group(t=3.92, P < 0.05). Cells apoptosis ratio of shRNA-XRCC2 group cells was the highest, reached(33.16±2.69)%, and there were significant differences compared with that of control(t=15.31, P < 0.01). Conclusion Knockdown of XRCC2 inhibited effectively the growth of colorectal cancer T84 cells in vitro. Silencing XRCC2 combined with radiation led to T84 cells arrested at G2/M phase and cells apoptosis and rendered T84 cells more sensitive to radiation. -
表 1 不同培养时间结肠癌T84细胞的生长抑制率(%,x±s)
Table 1. The growth inhibition rate at different culture time of colorectal cancer T84 cells(%, x±s)
培养时间(d) 抑制率 shRNA-SC shRNA-XRCC2 1 1.1±0.2 5.3±1.6 3 5.3±1.5 46.7±8.0 5 14.8±3.5 49.9±8.8 7 22.1±4.1 49.7±9.5 注:表中,XRCC2:X射线修复交叉互补基因2。 表 2 XRCC2基因沉默联合辐射后T84细胞的细胞周期和细胞凋亡率(%,x±s)
Table 2. The cell cycle distribution and cell apoptosis of T84 cells of silencing XRCC2 gene combined with radiation(%, x±s)
组别 细胞周期 细胞凋亡率 G0/G1 G2/M S 对照组 48.83±1.61 26.20±3.50 24.97±4.82 6.90±1.40 shRNA-SC组 67.58±1.16 24.64±1.38 7.78±0.42 7.76±2.15 shRXNRAC-C2组 41.13±7.85 38.51±4.15 20.36±11.47 33.16±2.69 注:表中,XRCC2:X射线修复交叉互补基因2。 -
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