CAJ | 학술논문

目的通过体外和体内实验研究结肠癌细胞在稳定转染Fas相关死亡结构域蛋白(FADD)基因后对5-氟尿嘧啶的敏感性,探讨FADD基因与5-氟尿嘧啶联合治疗结肠癌的可行性.方法 ①RT-PCR和Western印迹法检测稳定转染FADD基因的结肠癌细胞SW480/FADD、稳定转染空载体的结肠癌细胞SW480/neo和正常结肠癌细胞SW480中FADD基因的mRNA和蛋白表达水平.②MTT法检测3种细胞对5-氟尿嘧啶的敏感性.TUNEL法和流式双染法检测细胞凋亡率.Western印迹法检测caspase-8和caspase-3的表达.③裸鼠成瘤实验观察瘤体生长曲线,病理学和TUNEL法检测肿瘤细胞凋亡.结果 ①SW480/FADD细胞FADD基因mRNA和蛋白表达水平明显较SW480和SW480/neo增高(P＜0.05).②5-氟尿嘧啶对SW480/FADD的抑制率明显高于SW480和SW480/neo,差异有统计学意义(P＜0.05).③5-氟尿嘧啶(10 mg/L)干预48 h后,SW480/FADD凋亡率达(33.3±4.5)%,与SW480[(13.9±3.2)%]和SW480/neo[(14.1±3.4)%]间差异有统计学意义(P＜0.05).④5-氟尿嘧啶(10 mg/L)干预48 h后,SW480、SW480/neo的procaspase-8和procaspase-3表达比SW480/FADD增高,而cleaved easpase-8和cleaved caspase-3的表达比SW480/FADD降低(P＜0.05).⑤SW480/FADD对5-氟尿嘧啶更加敏感,瘤体体积增加幅度明显小于SW480和SW480/neo,差异有统计学意义(P＜0.05).结论稳定转染FADD基因可明显增加结肠癌细胞对5-氟尿嘧啶的敏感性,二者联合应用在结肠癌治疗中有潜在的价值.
목적 통과체외화체내실험연구결장암세포재은정전염Fas상관사망결구역단백(FADD)기인후대5-불뇨밀정적민감성,탐토FADD기인여5-불뇨밀정연합치료결장암적가행성.방법 ①RT-PCR화Western인적법검측은정전염FADD기인적결장암세포SW480/FADD、은정전염공재체적결장암세포SW480/neo화정상결장암세포SW480중FADD기인적mRNA화단백표체수평.②MTT법검측3충세포대5-불뇨밀정적민감성.TUNEL법화류식쌍염법검측세포조망솔.Western인적법검측caspase-8화caspase-3적표체.③라서성류실험관찰류체생장곡선,병이학화TUNEL법검측종류세포조망.결과 ①SW480/FADD세포FADD기인mRNA화단백표체수평명현교SW480화SW480/neo증고(P＜0.05).②5-불뇨밀정대SW480/FADD적억제솔명현고우SW480화SW480/neo,차이유통계학의의(P＜0.05).③5-불뇨밀정(10 mg/L)간예48 h후,SW480/FADD조망솔체(33.3±4.5)%,여SW480[(13.9±3.2)%]화SW480/neo[(14.1±3.4)%]간차이유통계학의의(P＜0.05).④5-불뇨밀정(10 mg/L)간예48 h후,SW480、SW480/neo적procaspase-8화procaspase-3표체비SW480/FADD증고,이cleaved easpase-8화cleaved caspase-3적표체비SW480/FADD강저(P＜0.05).⑤SW480/FADD대5-불뇨밀정경가민감,류체체적증가폭도명현소우SW480화SW480/neo,차이유통계학의의(P＜0.05).결론 은정전염FADD기인가명현증가결장암세포대5-불뇨밀정적민감성,이자연합응용재결장암치료중유잠재적개치.
Objective To examine the sensitivity of human colorectal carcinoma cells to 5-fluorouracil treatment by stable transfection of extrinsic Fas-associated death domain protein(FADD) gene，both in vitro and in vivo，so as to investigate the feasibility of combination therapy of FADD gene and 5-fluorouracil in human colorectal carcinoma. Methods ①RT-PCR and Western blotting were used to detect the expressions of both mRNA and protein of FADD gene in SW480／FADD (stably transfected with FADD)，SW480／neo and SW480 cells.②After treatment with 5-fluorouracil as an apoptotic inducer，in vitro cell growth activities were investigated by MTT assay.Cell apoptosis and its rates were determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL)assay and flow cytometry of annexin V-FITC／PI staining.The expressions of caspase-8 and caspase-3 were examined by Western blotting. ③To examine the inhibitory effect of FADD gene combined with 5-fluorouracil, tumor xenograft model was prepared for in vivo study.Results ① Compared with SW480 and SW480/neo cells, FADD mRNA and protein levels of SW480/FADD cells were higher (P<0.05). ② Inhibitory rate of SW480/FADD cells was remarkably higher than SW480 and SW480/neo cells (P＜0.05 ). ③ Forty-eight hours after treatment with 5-fluorouracil (10 mg/L), the apoptotic rate of SW480/FADD cells was (33.3 ± 4.5)%, which was higher than SW480 [(13. 9 ± 3. 2)%3 and SW480/neo [(14. 1 ± 3. 4)%], with significant difference (P< 0.05). ④ Forty-eight hours after treatment with 5-fluorouracil (10 mg/L),procaspase-8 and procaspase-3 expressions of SW480 and SW480/neo cells were higher than SW480/FADD cells, whereas their cleaved caspase-8 and cleaved caspase-3 expressions were lower than SW480/FADD cells (P＜0. 05).⑤ In in vivo study, SW480/FADD cells increased the efficacy of fluorouracil-induced inhibition of tumor growth in nude mice. Conclusions Stable overexpression of FADD increases sensitivity of the cells to 5-fluorouracil and combination of FADD with 5-fluorouracil will he a promising alternative in colorectal cancer treatment.