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肺癌是最常见的恶性肿瘤,其中,非小细胞肺癌(non-small cell lung cancer,NSCLC)是肺癌中最高发的(约占80%)。放疗是治疗NSCLC的一种主要治疗方式,但是,辐射抵抗是限制放疗被广泛应用的主要原因[1-2]。改变肿瘤细胞中特定的基因表达水平以影响肿瘤的放射敏感性是提高放疗疗效的一个有效方法,基因治疗与放疗联合使用会成为有前景的肿瘤治疗策略[3-4]。B7-H3蛋白于2001年首次被发现并被克隆,其是B7共刺激分子家族中的一员,具有调节T细胞介导的免疫反应的功能[5]。越来越多的研究揭示B7-H3蛋白可以抑制T细胞的增殖,减少其细胞因子的释放和转录因子的活化[6-7]。在正常组织中,B7-H3蛋白低表达[8]而在多种恶性肿瘤中都呈高表达,包括胰腺癌[9]、前列腺癌[10-11]、肾细胞癌[12-13]、NSCLC[14]等。但B7-H3蛋白在NSCLC中的临床意义和预后价值仍存在一定争议[15-17]。我们通过研究人为降低B7-H3基因表达对γ射线辐射诱导的人类肺癌A549细胞的凋亡和细胞周期的影响,探讨B7-H3在调节肺癌细胞放射敏感性中的作用。
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Western blotting法和实时定量PCR方法检测B7-H3蛋白和mRNA的改变,如图 1中A所示,与对照组相比,siB7-H3转染组细胞中的B7-H3蛋白水平明显降低。实时定量PCR结果经2-ΔΔCt分析后显示,siB7-H3转染组细胞中的siB7-H3 mRNA表达量也明显低于对照组细胞(图 1中B),且表达量的差异有统计学意义(t=-4.222,P=0.013)。这些实验结果表明,siB7-H3转染确实可以有效地降低siB7-H3蛋白和mRNA表达水平。
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如图 2所示,流式细胞仪分析结果显示,B7-H3表达水平的降低导致A549细胞的G0/G1期细胞阻滞,S和G2/M期细胞数量减少(图 2中B)。正如预期的,4 Gy γ射线照射后48 h,A549细胞出现轻微的G0/G1期阻滞和明显的G2/M期细胞阻滞(图 2中C)。而在siB7-H3转染的细胞,照射后不仅导致明显的G2/M周期阻滞,而且也有明显的G0/G1期阻滞(图 2中D)。
图 2 流式细胞仪检测siB7-H3对肺癌A549细胞的细胞周期的影响图中, A:对照组;B:siB7-H3转染组;C:照射组;D:照射+siB7-H3转染组。
Figure 2. Effects of siB7 -H3 on cell cycle progression of lung cancer A549 cells with flow cytometer. Un-irradiated (A) and 4Gy-irradiated (C) A549 cells without transfection; Un-irradiated (B) and 4Gy-irradiated (D) A549 cells transfected with siB7-H3.
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采用Annexin-V和PI双染色的流式细胞检测方法观察了B7-H3降低对A549细胞辐照引起细胞凋亡的影响。Annexin-V和PI双染色流式细胞检测典型结果解析为:左下为活细胞,左上为坏死细胞,而右象限均为凋亡细胞,其中右上象限为晚凋,右下为早凋(图 3)。如图 3所示,当对数生长的A549细胞受到4 Gy γ射线照射,细胞的坏死率(从2.59%上升至14.88%)和凋亡率(从7.73%上升至17.94%)明显增加(表 1)。siB7-H3转染组与对照组相比,降低B7-H3蛋白表达,A549细胞的坏死率和凋亡率无明显改变;照射+siB7-H3转染组与照射组相比,B7-H3蛋白的降低没有明显改变辐照诱导的细胞坏死率和凋亡率(图 3和表 1)。
图 3 流式细胞仪检测siB7-H3对肺癌A549细胞凋亡的影响图中, A:对照组;B:siB7-H3转染组;C:照射组;D:照射+siB7-H3转染组;AnnexinV-FITC:磷脂结合蛋白V-异硫氰酸荧光素;PI:碘化丙啶。
Figure 3. Effects of siB7-H3 on apoptosis of irradiated A549 cells with flow cytometer. Un-irradiated (A) and 4Gy-irradiated (C) A549 cells without transfection; Un-irradiated (B) and 4Gy-irradiated (D) A549 cells transfected with siB7-H3.
组别 活细胞率/% 坏死率/% 凋亡率/% 对照组 89.68 02.59 07.73 siB7-H3转染组 89.77 02.51 07.71 照射组 67.18 14.88 17.94 照射+ siB7-H3转染组 69.67 11.34 18.99 注:siB7-H3转染组为转染了靶向B7-H3蛋白的siRNA的肺癌A549细胞。 表 1 siB7-H3对肺癌A549细胞凋亡的影响
Table 1. Effects of siB7-H3 on apoptosis of irradiated lung cancer
降低B7-H3蛋白对受照肺癌细胞A549细胞周期和凋亡的影响
Study on the effects of B7-H3 down-regulation on cell cycle and apoptosis of lung cancer A549 cells
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摘要:
目的观察用小干扰RNA(siRNA)敲降B7-H3蛋白的表达对人肺癌细胞A549的细胞周期和凋亡的影响。 方法培养人肺癌A549细胞,将B7-H3蛋白siRNA瞬时转染于A549细胞(称为siB7-H3转染组)。实验分为4组,即对照组、siB7-H3转染组、照射组、照射+siB7-H3转染组。采用137Cs γ射线一次性照射,照射剂量为4 Gy;用Western blotting法和实时定量PCR分别检测B7-H3蛋白和mRNA的表达;流式细胞仪检测细胞周期和细胞凋亡的改变。 结果与对照组相比,siB7-H3转染组的B7-H3蛋白水平明显降低,mRNA表达量也明显低于对照组且差异有统计学意义(t=-4.222,P=0.013)。siB7-H3转染组细胞的G0/G1期细胞阻滞,S和G2/M期细胞数量减少;与对照组相比,照射组出现轻微的G0/G1期阻滞和明显的G2/M期细胞阻滞,照射+siB7-H3转染组的G0/G1、G2/M期均有明显的阻滞。照射后48 h,与对照组相比,照射组细胞的坏死率和凋亡率明显升高,siB7-H3转染组和照射+siB7-H3转染组的坏死率和凋亡率均无明显改变。 结论降低肺癌A549细胞中B7-H3蛋白表达水平可明显增加辐射诱导的G0/G1期阻滞,从而提示B7-H3表达水平的改变可能通过调节G0/G1细胞周期检查点而对肺癌细胞放射敏感性产生重要的调节功能。 Abstract:ObjectiveTo explore the effects of B7-H3 down-regulation on cell cycle and apoptosis in lung cancer A549 cells. Method Human lung cancer cell line A549 were cultured. siB7-H3 RNA which can specifically silence the expression of B7-H3 protein was transfected into A549 cells. 137Cs γ-ray irradiation was used with a single dose of 4 Gy. Experiments included control group, siB7-H3 transfected group, irradiation group, and irradiation+siB7-H3 transfected group. After transfected with siB7-H3, Western blotting and quantitative real-time PCR assays were used to detect the expression of B7-H3 protein and mRNA in A549 cells. The cell cycle and apoptosis of A549 cells following 4 Gy irradiation were detected by flow cytometry. ResultsThe protein and mRNA expression of B7-H3 were significantly decreased in A549 cells transfected with siB7-H3 compared with the control group, and the differences between the two groups were statistically significant(t=-4.222, P=0.013). siB7-H3 transfection induced significant effect on cell cycle with increase of G0/G1 phase arrest and reduction of S and G2/M phase population. A mild enhanced G0/G1 phase arrest and an obvious enhanced G2/M phase arrest of irradiation group were detected compared with the control group. An enhanced G0/G1 and G2/M phase arrest of irradiation+siB7-H3 transfected group were detected compared with the control group. Compared with the control group, the necrosis and apoptosis induction of the irradiated group significantly increased at 48 h after irradiation. However, No significant alterations of necrosis and apoptosis induction were observed between irradiation group and irradiation+ siB7-H3 transfected group. ConclusionsDown-regulation of B7-H3 can significantly elevated the G0/G1 arrest in A549 cells following radiation. This conclusion indicated that the alteration of B7-H3 expression could play a key role in the regulation of the radiosensitivity of lung cancer via mediating the G0/G1 check point. -
Key words:
- Lung neoplasms /
- Carcinoma, non-small-cell lung /
- Cell cycle /
- Apoptosis /
- B7-H3 /
- siRNA
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图 2 流式细胞仪检测siB7-H3对肺癌A549细胞的细胞周期的影响图中, A:对照组;B:siB7-H3转染组;C:照射组;D:照射+siB7-H3转染组。
Figure 2. Effects of siB7 -H3 on cell cycle progression of lung cancer A549 cells with flow cytometer. Un-irradiated (A) and 4Gy-irradiated (C) A549 cells without transfection; Un-irradiated (B) and 4Gy-irradiated (D) A549 cells transfected with siB7-H3.
图 3 流式细胞仪检测siB7-H3对肺癌A549细胞凋亡的影响图中, A:对照组;B:siB7-H3转染组;C:照射组;D:照射+siB7-H3转染组;AnnexinV-FITC:磷脂结合蛋白V-异硫氰酸荧光素;PI:碘化丙啶。
Figure 3. Effects of siB7-H3 on apoptosis of irradiated A549 cells with flow cytometer. Un-irradiated (A) and 4Gy-irradiated (C) A549 cells without transfection; Un-irradiated (B) and 4Gy-irradiated (D) A549 cells transfected with siB7-H3.
表 1 siB7-H3对肺癌A549细胞凋亡的影响
Table 1. Effects of siB7-H3 on apoptosis of irradiated lung cancer
组别 活细胞率/% 坏死率/% 凋亡率/% 对照组 89.68 02.59 07.73 siB7-H3转染组 89.77 02.51 07.71 照射组 67.18 14.88 17.94 照射+ siB7-H3转染组 69.67 11.34 18.99 注:siB7-H3转染组为转染了靶向B7-H3蛋白的siRNA的肺癌A549细胞。 -
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