小牛脾提取物注射液对人肺癌细胞增殖 及放射敏感性的影响

Effect of calf spleen extraction injection on proliferation and radiosensitivity in lung cancer cell

摘要:

目的 探讨小牛脾提取物注射液（CSEI）对人肺癌细胞A549 增殖及放射敏感性的影响。 方法 分别采用不同浓度的CSEI 及不同放射剂量处理A549 细胞48 h 后，用MTT 法和平板克隆实验检测 细胞增殖抑制率、增敏比（SER）及细胞集落形成率；PI 单染和Annexin V-FITC/PI 双染法检测CSEI 和 放射线对细胞周期和凋亡的影响；彗星实验检测细胞核DNA双链损伤；Western blotting检测Bcl-2、Bax、MDR1 及Survivin 的表达。结果 经MTT 法检测，1.0 mg/ml 是CESI 的无毒最高剂量。1.0 mg/ml CESI 预处理后， 可增加不同剂量（2、4、6、8 及10 Gy）放射线对A549 细胞增殖的抑制作用（P <0.05），SER 为（1.42±0.06）。 克隆形成实验显示，放射线（5 Gy）+CSEI（1.0 mg/ml）组集落形成能力最弱（P <0.05）。彗星实验和流式 细胞术结果显示，放射线（5 Gy）+CSEI（1.0 mg/ml）组细胞核拖尾细胞数、尾长、尾距及凋亡率多于空白 对照组、放射线组及CSEI 组细胞（P <0.05），而且与空白对照组比较，放射线（5 Gy）+CSEI（1.0 mg/ml） 组G1 期细胞和S 期细胞减少，而G2/M 期细胞增多；同时放射线（5 Gy）+CSEI（1.0 mg/ml）组Bcl-2 和 Survivin 蛋白表达下降，Bax 蛋白表达升高（P <0.05）。结论 1.0 mg/ml CSEI 可增强人肺腺癌细胞系A549 对放射线的敏感性，其作用机制可能与影响肿瘤细胞周期、促进肿瘤细胞凋亡及细胞核DNA 双链损伤等 有关。

Abstract:

Objective To discuss the effect of calf spleen extraction injection (CSEI) on proliferation and radiosensitivity in lung cancer cell A549. Methods A549 cells were treated with different concentrations of CSEI or different radiation doses for 48h. Cell proliferation inhibition rate, sensitivity enhancement ratio (SER) and colony formation rate were accessed by MTT assay and plate colony formation assay. Propidium iodide (PI) staining and Annexin V-FITC/PI staining were respectively used for cell cycle and apoptosis of A549 cells. Comet assay was to detected the DNA damage. The B-cell lymphoma-2 (Bcl-2), bcl-2-associated X protein (Bax), multi-drug resistance1 (MDR1) and Survivin proteins levels of A549 cells were detected by western blot. Results By MTT assay, 1.0 mg/mL would be the highest non-toxic dose of CSEI. CSEI (1.0 mg/mL) pretreatment might increase the inhibition rate of radiation (2, 4, 6, 8, 10 Gy) on A549 cells (P < 0.05). And sensitivity enhancement ratio (SER) was (1.42±0.06). Cloning formation experiment showed the colony formation ability of CSEI (1.0 mg/mL) + radiation (5 Gy) group was the least (P < 0.05). As the results of comet assay and flow cytometry, the tailing cell amount, tail length, tail distance and apoptosis rate of A549 cells in CSEI (1.0 mg/mL) + radiation (5 Gy) group were more than those in control group, radiation group and CSEI group (P < 0.05). Meanwhile, compared with control group, radiation group and CSEI group, cell amount in G1 stage and S stage were less, but in G2/M stages was more; and Bcl-2 and Survivin proteins levels were lower, Bax protein level was higher (P < 0.05). Conclusions There are significant evidences that 1.0 mg/mL of CESI would enhance the sensitivity of A549 cells to radiation, which could be related with tumor cell cycle, apoptosis, nucleus DNA double-strand damage, etc.