中华普通外科杂志
中華普通外科雜誌
중화보통외과잡지
CHINESE JOURNAL OF GENERAL SURGERY
2013年
2期
129-133
,共5页
侯庆生%丁谓%陈德喜%张玉林%郭洪亮
侯慶生%丁謂%陳德喜%張玉林%郭洪亮
후경생%정위%진덕희%장옥림%곽홍량
结直肠肿瘤%肿瘤抑制蛋白质,p53%自噬%细胞凋亡
結直腸腫瘤%腫瘤抑製蛋白質,p53%自噬%細胞凋亡
결직장종류%종류억제단백질,p53%자서%세포조망
Colorectal neoplasms%Tumor suppressor protein,p53%Autophagy%Apoptosis
目的 探讨p53凋亡刺激蛋白2(apoptosis stimulating protein 2 0f p53,ASPP2)对饥饿诱导的大肠癌HCT116 p53-/-(p53缺失)细胞自噬和凋亡的影响.方法 实验分3组:(1)绿色荧光蛋白腺病毒(green fluorescent protein adenovirus,rAd-GFP)感染组(对照组)；(2)自噬抑制剂LY294002处理组；(3) ASPP 2腺病毒(ASPP2 adenovirus,rAd-ASPP2)感染组.无血清培养基分别培养0、24、48 h诱导细胞自噬和凋亡.利用calcein/PI吸收试验观察各组细胞凋亡水平.细胞转染红色荧光蛋白自噬质粒Lc3(cerise fluorescent protein autophagy plasmid Lc3,CFP-Lc3),在荧光显微镜下观察各组细胞自噬水平.结果 rAd-GFP感染组饥饿24h细胞的自噬水平升高显著(0 h:1.04±0.24；24 h:12.17 ±0.86,P＜0.05),而凋亡水平改变差异无统计学意义(0 h:2.01％±0.06％；24 h:3.23％±0.34％,P＞0.05)；饥饿时间延长至48 h,自噬水平(0 h:1.04±0.24；48 h:21.09±3.32)和凋亡水平(0h:2.01％±0.06％；48 h:30.20％±3.18％)升高,差异均具有统计学意义(均P＜0.05).使用LY294002抑制细胞自噬,饥饿24 h细胞凋亡显著增多(rAd-GFP组:3.23％±0.34％；LY294002组:15.68％±1.24％,P＜0.01),而饥饿48 h凋亡显著减少(rAd-GFP组:30.20％±3.18％；LY294002组:25.44％±3.01％,P ＜0.05).rAd-ASPP2感染组饥饿24 h自噬显著降低(rAd-GFP组:12.17±0.86;ASPP2组:1.45±0.45,P＜0.01),而凋亡显著升高(rAd-GFP组:3.23％±0.34％；ASPP2组:10.45％ ±0.81％,P＜0.05)；饥饿48 h自噬(rAd-GFP组:21.09 ±3.32；ASPP2组:29.93±3.48)和凋亡(rAd-GFP组:30.20％±3.18％；ASPP2组:36.72％±2.74％)水平均显著升高(均P＜0.05).结论 ASPP2通过对自噬的双向调节促进饥饿诱导的大肠癌HCT116 p53-/-细胞凋亡.
目的 探討p53凋亡刺激蛋白2(apoptosis stimulating protein 2 0f p53,ASPP2)對饑餓誘導的大腸癌HCT116 p53-/-(p53缺失)細胞自噬和凋亡的影響.方法 實驗分3組:(1)綠色熒光蛋白腺病毒(green fluorescent protein adenovirus,rAd-GFP)感染組(對照組)；(2)自噬抑製劑LY294002處理組；(3) ASPP 2腺病毒(ASPP2 adenovirus,rAd-ASPP2)感染組.無血清培養基分彆培養0、24、48 h誘導細胞自噬和凋亡.利用calcein/PI吸收試驗觀察各組細胞凋亡水平.細胞轉染紅色熒光蛋白自噬質粒Lc3(cerise fluorescent protein autophagy plasmid Lc3,CFP-Lc3),在熒光顯微鏡下觀察各組細胞自噬水平.結果 rAd-GFP感染組饑餓24h細胞的自噬水平升高顯著(0 h:1.04±0.24；24 h:12.17 ±0.86,P＜0.05),而凋亡水平改變差異無統計學意義(0 h:2.01％±0.06％；24 h:3.23％±0.34％,P＞0.05)；饑餓時間延長至48 h,自噬水平(0 h:1.04±0.24；48 h:21.09±3.32)和凋亡水平(0h:2.01％±0.06％；48 h:30.20％±3.18％)升高,差異均具有統計學意義(均P＜0.05).使用LY294002抑製細胞自噬,饑餓24 h細胞凋亡顯著增多(rAd-GFP組:3.23％±0.34％；LY294002組:15.68％±1.24％,P＜0.01),而饑餓48 h凋亡顯著減少(rAd-GFP組:30.20％±3.18％；LY294002組:25.44％±3.01％,P ＜0.05).rAd-ASPP2感染組饑餓24 h自噬顯著降低(rAd-GFP組:12.17±0.86;ASPP2組:1.45±0.45,P＜0.01),而凋亡顯著升高(rAd-GFP組:3.23％±0.34％；ASPP2組:10.45％ ±0.81％,P＜0.05)；饑餓48 h自噬(rAd-GFP組:21.09 ±3.32；ASPP2組:29.93±3.48)和凋亡(rAd-GFP組:30.20％±3.18％；ASPP2組:36.72％±2.74％)水平均顯著升高(均P＜0.05).結論 ASPP2通過對自噬的雙嚮調節促進饑餓誘導的大腸癌HCT116 p53-/-細胞凋亡.
목적 탐토p53조망자격단백2(apoptosis stimulating protein 2 0f p53,ASPP2)대기아유도적대장암HCT116 p53-/-(p53결실)세포자서화조망적영향.방법 실험분3조:(1)록색형광단백선병독(green fluorescent protein adenovirus,rAd-GFP)감염조(대조조)；(2)자서억제제LY294002처리조；(3) ASPP 2선병독(ASPP2 adenovirus,rAd-ASPP2)감염조.무혈청배양기분별배양0、24、48 h유도세포자서화조망.이용calcein/PI흡수시험관찰각조세포조망수평.세포전염홍색형광단백자서질립Lc3(cerise fluorescent protein autophagy plasmid Lc3,CFP-Lc3),재형광현미경하관찰각조세포자서수평.결과 rAd-GFP감염조기아24h세포적자서수평승고현저(0 h:1.04±0.24；24 h:12.17 ±0.86,P＜0.05),이조망수평개변차이무통계학의의(0 h:2.01％±0.06％；24 h:3.23％±0.34％,P＞0.05)；기아시간연장지48 h,자서수평(0 h:1.04±0.24；48 h:21.09±3.32)화조망수평(0h:2.01％±0.06％；48 h:30.20％±3.18％)승고,차이균구유통계학의의(균P＜0.05).사용LY294002억제세포자서,기아24 h세포조망현저증다(rAd-GFP조:3.23％±0.34％；LY294002조:15.68％±1.24％,P＜0.01),이기아48 h조망현저감소(rAd-GFP조:30.20％±3.18％；LY294002조:25.44％±3.01％,P ＜0.05).rAd-ASPP2감염조기아24 h자서현저강저(rAd-GFP조:12.17±0.86;ASPP2조:1.45±0.45,P＜0.01),이조망현저승고(rAd-GFP조:3.23％±0.34％；ASPP2조:10.45％ ±0.81％,P＜0.05)；기아48 h자서(rAd-GFP조:21.09 ±3.32；ASPP2조:29.93±3.48)화조망(rAd-GFP조:30.20％±3.18％；ASPP2조:36.72％±2.74％)수평균현저승고(균P＜0.05).결론 ASPP2통과대자서적쌍향조절촉진기아유도적대장암HCT116 p53-/-세포조망.
Objective To investigate the role of ASPP2 (apoptosis stimulating protein 2 of p53,ASPP2) in starvation-induced autophagy and apoptosis of colorectal cancer HCT116 p53-/-(p53 gene deletion) cell line.Methods The study included three experiment groups:green fluorescent protein adenovirus (rAd-GFP) infection group,autophagy inhibitor LY294002 treatment group and ASPP2 adenovirus (rAd-ASPP2) infection group.Celluar autophagy and apoptosis were induced by coculturing with serum-free medium for 0 h,24 h,48 h.Apoptosis level was detected by Calcein/PI uptaking test.Autophagy level was observed under the fluorescence microscope via transfection with cerise fluorescent protein autophagy plasmid CFP-Lc3.Results In control group,starvation for 24 hours significantly promoted autophagy of HCT116 cells (0 h:1.04 ±0.24; 24 h:12.17 ±0.86,P ＜0.05),while apoptosis was not increased (0 h:2.01％ ±0.06％; 24 h:3.23％ ±0.34％,P ＞0.05).With 48 h starvation,autophagy(0 h:1.04 ±0.24; 48 h:21.09 ±3.32) and apoptosis(0 h:2.01％ ±0.06％ ; 48 h:30.20％ ±3.18％)of HCT116 increased (P ＜ 0.05).With the use of LY294002 apoptosis induced by 24 h starvation significantly increased (rAd-GFP group:3.23％ ± 0.34％ ; LY294002 group:15.68％ ± 1.24％,P ＜0.01),but aopotosis under 48 h starvation decreased (rAd-GFP group:30.20％ ± 3.18％; LY294002group:25.44％ ± 3.01％,P ＜ 0.05).With ASPP2 transfection,autophagy under 24 h starvation significantly declined (rAd-GFP group:12.17 ± 0.86,ASPP2 group:1.45 ± 0.45,P ＜ 0.01),and apoptosis increased(rAd-GFP group:3.23％ ± 0.34％ ; ASPP2 group:10.45％ ± 0.81％,P ＜ 0.05).Both autophagy (rAd-GFP group:21.09 ± 3.32; ASPP2 group:29.93 ± 3.48) and apoptosis (rAd-GFP group:30.20％ ±3.18％ ; ASPP2 group:36.72％ ±2.74％) were higher than that in controls under 48 h starvation (P ＜ 0.05).Conclusions ASPP2 probably promotes apoptosis of colorectal cancer cells by two-way regulated autophagy.
