CAJ | 학술논문

目的:研究结肠癌K-ras基因突变导致对西妥昔单抗耐药的可能机制.方法:通过基因测序及免疫细胞化学染色,筛选表皮生长因子受体(EGFR)表达阳性,同时K-ras基因呈突变型及野生型的结肠癌细胞各1株,应用细胞增殖实验(CCK8方法)、流式细胞技术(Annexin V标记),检测西妥昔单抗(C225)在体外对结肠癌细胞增殖和凋亡的影响.同时用K-ras呈野生型和突变型的结肠癌细胞株,分别建立裸鼠皮下移植瘤模型,分成HT29-C225、HT29-NS、SW620-C225及SW620-NS组,给予西妥昔单抗(每3天注射1次,每次0.5 mL,共4周).治疗后,绘制肿瘤生长曲线,用TDT介导的缺口末端标记技术(TUNEL)检测细胞凋亡,用定量PCR、免疫组织化学染色(IHC)及蛋白印迹(Western-blot)等技术检测移植瘤标本中EGFR信号转导通路中AKT及其磷酸化蛋白的表达.结果:体外增殖及凋亡实验显示,西妥昔单抗对不同K-ras基因型的结肠癌细胞均未能显示细胞毒作用.而体内研究发现.西妥昔单抗能明显抑制K-ras野生型结肠癌裸鼠皮下移植瘤的生长,但对K-ras突变型者抑制作用较差.对K-ras突变型结肠癌裸鼠皮下移植瘤的定量PCR检测结果显示,西妥昔单抗治疗组裸鼠的AKT基因表达率高于对照组.IHC及Western-blot显示,西妥昔单抗治疗组与对照组间AKT蛋白的表达率无明显差异,但西妥昔单抗治疗组裸鼠的p-AKT表达率高于对照组.结论:K-ras突变状态与西妥昔单抗的疗效相关,K-ras突变型者EGFR信号通路中的衔接蛋白AKT呈自主激活状态,该基因突变可能是导致对西妥昔单抗耐药的机制之一.
목적:연구결장암K-ras기인돌변도치대서타석단항내약적가능궤제.방법:통과기인측서급면역세포화학염색,사선표피생장인자수체(EGFR)표체양성,동시K-ras기인정돌변형급야생형적결장암세포각1주,응용세포증식실험(CCK8방법)、류식세포기술(Annexin V표기),검측서타석단항(C225)재체외대결장암세포증식화조망적영향.동시용K-ras정야생형화돌변형적결장암세포주,분별건립라서피하이식류모형,분성HT29-C225、HT29-NS、SW620-C225급SW620-NS조,급여서타석단항(매3천주사1차,매차0.5 mL,공4주).치료후,회제종류생장곡선,용TDT개도적결구말단표기기술(TUNEL)검측세포조망,용정량PCR、면역조직화학염색(IHC)급단백인적(Western-blot)등기술검측이식류표본중EGFR신호전도통로중AKT급기린산화단백적표체.결과:체외증식급조망실험현시,서타석단항대불동K-ras기인형적결장암세포균미능현시세포독작용.이체내연구발현.서타석단항능명현억제K-ras야생형결장암라서피하이식류적생장,단대K-ras돌변형자억제작용교차.대K-ras돌변형결장암라서피하이식류적정량PCR검측결과현시,서타석단항치료조라서적AKT기인표체솔고우대조조.IHC급Western-blot현시,서타석단항치료조여대조조간AKT단백적표체솔무명현차이,단서타석단항치료조라서적p-AKT표체솔고우대조조.결론:K-ras돌변상태여서타석단항적료효상관,K-ras돌변형자EGFR신호통로중적함접단백AKT정자주격활상태,해기인돌변가능시도치대서타석단항내약적궤제지일.
Objective To investigate the possible mechanism of K-ras mutation induced resistance to cetuximab. Methods Two colon cancer cell lines, HT29 and SW620, which were epidermal growth factor receptor (EGFR) positive, but separately with K-ras wild type and mutant type. were screened. The cytotoxic effect and apoptosis-inducing effect of cetuximab in vitro were evaluated via the cell proliferation assay (CCK8) and flow cytomety (Annexin V labeled). Subcutaneous xenograft nude mice model was established, and divided into 4 groups : HT29-C225, HT29-NS, SW620-C225 and SW620-NS; the inhibitory effects of cetuximab on these 2 colon cancer cell lines in vivo were observed. The EGFR expression, K-ras status. its down-stream signal protein AKT and its phospho-protein in EGFR pathway were detected by immunohistochemical staining and Westem-blot. Results To colon cancer xenograft models, cetuximab significantly inhibited the growth of colon cancer xenograft with the K-ras wild-type; yet, in colon cancer xenograft with the K-ras mutant-type, cetuximab did not inhibit the tumor growth. Detailed molecular detection demonstrated that the expression of phospho-AKT was elevated in the K-ras mutant colon cancer xenograft. Conclusions The K-ras status might influence the effects of cetuximab treatment. K-rass mutation might lead to the spontaneous activation of its down-stream signal adaptor protein AKT, thus in turn trigger the EGFR pathway activation and cause cetuximab resistance.