EGFR单克隆抗体影响结肠癌细胞生物学行为的实验研究

The experimental research on the biology behavior of colon cancer cell influenced by EGFR monoclonal antibody

背景与目的:表皮生长因子受体(epidermal growth factor receptor,EGFR)在多种实体肿瘤中存在过表达。本研究旨在探讨EGFR单克隆抗体西妥昔单抗(cetuximab)对体外培养的人结肠癌细胞株HT29增殖和凋亡的影响,并初步探讨其作用机制。方法:不同浓度EGFR单克隆抗体(12.5、25、50、100μg/mL)作用于对数生长期的HT29细胞,倒置显微镜下观察细胞生长情况,四甲基偶氮唑蓝(MTT)法观察EGFR单克隆抗体对HT29细胞生长的抑制作用;流式细胞术(flow cytometry,FCM)检测对细胞周期和细胞凋亡的影响;免疫细胞化学(SP)法测定细胞内Bcl-2、Caspase-3蛋白表达变化。结果:MTT比色法显示不同浓度的EGFR单克隆抗体可抑制HT29细胞增殖,抑制作用呈时间和剂量依赖性,试验组与对照组及各试验组之间比较差异均有统计学意义(P<0.05)。FCM检测显示,HT29经EGFR单克隆抗体作用48 h后细胞周期有明显变化,G0/G1期细胞比例上升,S期细胞比例下降,呈浓度依赖性(P<0.05),细胞凋亡率与EGFR单克隆抗体浓度呈正相关。SP法显示,HT29细胞Caspase-3蛋白随EGFR单克隆抗体浓度增加而表达增加,Bcl-2蛋白随药物浓度增加表达下降(P<0.05)。结论:EGFR单克隆抗体可抑制人结肠癌细胞HT29增殖,诱导细胞凋亡,其作用可能与上调Caspase-3蛋白表达、下调Bcl-2蛋白表达有关。

Background and purpose： Epidermal growth factor receptor （EGFR） over-expressed in kinds of different malignant tumor. This study aimed to investigate the roll of epidermal growth factor receptor inhibitor （Cetuximab） in the human colon carcinoma cell HT29. Methods： Four groups treated with different concentrations （12.5, 25, 50 and 100 μg/mL） of Cetuximab were assayed by MTT testing to compare the cell growth. The cell cycles and apoptosis were detected by the flow cytometry （FCM）. The expressions of bcl-2 and Caspase-3 protein were performed by imrnunohistochemistry. Results： MTT assay indicated that Cetuximab inhibited the proliferation of HT29 cell in dose-and-time dependent manner （P〈0.05）. The cell apoptosis was observed after Cetuximab treating 48 hours, and the ratio of G0/Gt phase was increased but the ratio of S phase was decreased in dose-dependent manner （P〈0.05）. Immunohistochemistry results showed that the expression of the Caspase-3 protein were upregulated while the bcl-2 protein was downregulated after Cetuximab treating 48 hours （P〈0.05）. Conclusion： The proliferation of HT29 cell was inhibited by Cetuximab and in dose-and-time dependent manner. Moreover, Cetuximab arrested cell cycle at G0/GI phase and induced cell apoptosis.