CAJ | 학술논문

目的探讨表皮生长因子受体(EGFR)表达及其下游基因K-ras、B-raf和PIK3CA突变对结直肠癌细胞放射敏感性的影响.方法对9株人结直肠癌细胞株应用实时定量RT-PCR检测EGFR表达,并检测K-ras、B-raf和PIK3CA基因的突变状态；对各细胞株行梯度剂量射线照射后,行克隆形成实验明确其放射敏感性、行Hoechst 33258染色凋亡形态学观察和流式细胞术检查细胞凋亡及细胞周期.结果人结直肠癌细胞株EGFR表达与放疗抵抗呈正相关(r=0.717,P=0.030),PIK3CA突变与放疗敏感性有关(t=2.401,P=0.047),K-ras和B-raf突变与放疗敏感性无关(均P＞0.05).放疗敏感细胞株(HCT116)总凋亡率在低放射剂量(2 Gy)时即显著增加,并随放射剂量增加而继续增加(P＜0.05),而放疗抵抗细胞株(HT29)总凋亡率只有放射剂量较大时(6Gy)才明显增加.G1/G0期HCT116细胞株比例随放疗剂量增加显著降低(P＜0.05),而HT29细胞株G1/G0期比例在放疗剂量增加时无明显变化(P＞0.05).结论 EGFR通路中多个位点同时参与了结直肠癌细胞的放疗抵抗或敏感作用,EGFR高表达与放疗抵抗相关,PIK3CA突变与放疗敏感有关,放疗抵抗机制可能与抑制细胞凋亡和增加细胞在G1/G0期停顿相关.
목적 탐토표피생장인자수체(EGFR)표체급기하유기인K-ras、B-raf화PIK3CA돌변대결직장암세포방사민감성적영향.방법 대9주인결직장암세포주응용실시정량RT-PCR검측EGFR표체,병검측K-ras、B-raf화PIK3CA기인적돌변상태；대각세포주행제도제량사선조사후,행극륭형성실험명학기방사민감성、행Hoechst 33258염색조망형태학관찰화류식세포술검사세포조망급세포주기.결과 인결직장암세포주EGFR표체여방료저항정정상관(r=0.717,P=0.030),PIK3CA돌변여방료민감성유관(t=2.401,P=0.047),K-ras화B-raf돌변여방료민감성무관(균P＞0.05).방료민감세포주(HCT116)총조망솔재저방사제량(2 Gy)시즉현저증가,병수방사제량증가이계속증가(P＜0.05),이방료저항세포주(HT29)총조망솔지유방사제량교대시(6Gy)재명현증가.G1/G0기HCT116세포주비례수방료제량증가현저강저(P＜0.05),이HT29세포주G1/G0기비례재방료제량증가시무명현변화(P＞0.05).결론 EGFR통로중다개위점동시삼여료결직장암세포적방료저항혹민감작용,EGFR고표체여방료저항상관,PIK3CA돌변여방료민감유관,방료저항궤제가능여억제세포조망화증가세포재G1/G0기정돈상관.
Objective To investigate the effect of epithermal growth factor receptor (EGFR) expression and K-ras,B-raf and PIK3CA mutation status on the radiosensitivity of human colorectal carcinoma (CRC) cell lines in vitro.Methods Real-time RT-PCR was used to measure EGFR mRNA expression in nine human CRC cell lines,and K-ras,B-raf and PIK3CA mutation status of each CRC cell line was also identified respectively.After treatment with irradiation at graded dose,the cell viability was measured by clonogenic survival assay.The rate of cell apoptosis and cell cycle distribution were tested by flow cytometry.The cell morphology was observed with hoechst 33258 staining to analyze the correlation between EGFR mRNA expression and radiosensitivity of CRC cell lines.Results A positive correlation between EGFR mRNA expression and survival fraction of 2 Gy (SF2) was observed(r=0.717,P=0.030).Association was also identified between the mutation status of PIK3CA and radiosensitivity (t=2.401,P=0.047),while mutation status of K-ras and B-raf was not associated with radiosensitivity.At 48-hour after exposing to irradiation,the apoptosis rate of radiosensitive cell line (HCT116) was significantly increased in a dose-dependent manner(P＜0.05),while the apoptosis rate of radioresistant cell line(HT29) was significantly increased only when radiation dose increased to 6 Gy.The ratio of G0/G1 phase was reduced significantly with the increase of radiation dose in radiosensitive cell line (HCT116,P＜0.05),while this trend was not observed in radioresistant cell line(HT29,P＞0.05).Conclusions Over-expression of EGFR mRNA is correlated to radioresistance of human CRC cell lines,and mutation status of PIK3CA is closely related with radiosensitivity of CRC cells.The inhibition of apoptosis and G0/G1 arrest may induce the radioresistance of CRC cell lines.