中华烧伤杂志
中華燒傷雜誌
중화소상잡지
16
2010年
2期
128-132
,共5页
曹永倩%王法刚%霍然%蔡景龙%冯永强%李强%王一兵
曹永倩%王法剛%霍然%蔡景龍%馮永彊%李彊%王一兵
조영천%왕법강%곽연%채경룡%풍영강%리강%왕일병
寡核苷酸类,反义%黑色素瘤%半胱氨酸天冬氨酸蛋白酶3%存活素
寡覈苷痠類,反義%黑色素瘤%半胱氨痠天鼕氨痠蛋白酶3%存活素
과핵감산류,반의%흑색소류%반광안산천동안산단백매3%존활소
Oligonucleotides,antisense%Melanoma%Caspase-3%Survivin
目的 了解存活素反义寡脱氧核苷酸(ASODN)对人恶性黑色素瘤细胞(hMMC)增殖和凋亡的影响.方法 取对数生长期hMMC株A375,按随机数字表法分为对照组(不转染)、正义链组(转染600 nmol/L存活素正义寡脱氧核苷酸)、错义链组(转染600 nmol/L存活素错义寡脱氧核苷酸)、脂质体组(仅用脂质体处理)、反义链组(转染存活素ASODN,根据转染物浓度再分为200、400、600 nmol/L 3个亚组),倒置荧光显微镜下观察转染效果.采用噻唑蓝法测定细胞存活情况并计算细胞增殖抑制率,双变量流式细胞仪检测细胞凋亡率及细胞周期,蛋白质印迹法检测存活素蛋白的表达,激酶法检测半胱氨酸天冬氨酸蛋白酶3(caspase-3)的活性.对数据进行方差分析.结果 (1)正义链组、错义链组、反义链600 nmol/L组细胞转染率均大于80%.(2)反义链200、400、600nmol/L组转染后24 h细胞增殖抑制率[(10.30±0.56)%、(16.69±0.58)%、(24.67±0.67)%]较正义链组[(5.23±0.25)%]、错义链组[(5.09±0.13)%]、脂质体组[(4.70±0.45)%]显著增加(F=746.91,P值均小于0.05),且随转染时间延长,增殖抑制率增加明显.(3)反义链200、400、600nmol/L组转染后24 h细胞凋亡率分别为(13.5±1.9)%、(20.1±1.5)%、(32.1±2.9)%,显著高于对照组、正义链组、错义链组、脂质体组的(6.5±0.6)%、(5.6±0.7)%、(6.4±1.0)%、(6.5±1.3)%(F=139.9,P值均小于0.05),细胞被阻滞在G2/M期.(4)与对照组比较,反义链各浓度组存活素蛋白表达量减少,caspase-3活性明显增高(F=63.1,P值均小于0.05);正义链组、错义链组、脂质体组caspase-3活性与对照组比较,差异无统计学意义(F=0.512,P值均大于0.05).结论 存活素ASODN能够呈浓度-时间依赖性抑制hMMC株A375增殖,诱导G2/M期阻滞,促进其凋亡.
目的 瞭解存活素反義寡脫氧覈苷痠(ASODN)對人噁性黑色素瘤細胞(hMMC)增殖和凋亡的影響.方法 取對數生長期hMMC株A375,按隨機數字錶法分為對照組(不轉染)、正義鏈組(轉染600 nmol/L存活素正義寡脫氧覈苷痠)、錯義鏈組(轉染600 nmol/L存活素錯義寡脫氧覈苷痠)、脂質體組(僅用脂質體處理)、反義鏈組(轉染存活素ASODN,根據轉染物濃度再分為200、400、600 nmol/L 3箇亞組),倒置熒光顯微鏡下觀察轉染效果.採用噻唑藍法測定細胞存活情況併計算細胞增殖抑製率,雙變量流式細胞儀檢測細胞凋亡率及細胞週期,蛋白質印跡法檢測存活素蛋白的錶達,激酶法檢測半胱氨痠天鼕氨痠蛋白酶3(caspase-3)的活性.對數據進行方差分析.結果 (1)正義鏈組、錯義鏈組、反義鏈600 nmol/L組細胞轉染率均大于80%.(2)反義鏈200、400、600nmol/L組轉染後24 h細胞增殖抑製率[(10.30±0.56)%、(16.69±0.58)%、(24.67±0.67)%]較正義鏈組[(5.23±0.25)%]、錯義鏈組[(5.09±0.13)%]、脂質體組[(4.70±0.45)%]顯著增加(F=746.91,P值均小于0.05),且隨轉染時間延長,增殖抑製率增加明顯.(3)反義鏈200、400、600nmol/L組轉染後24 h細胞凋亡率分彆為(13.5±1.9)%、(20.1±1.5)%、(32.1±2.9)%,顯著高于對照組、正義鏈組、錯義鏈組、脂質體組的(6.5±0.6)%、(5.6±0.7)%、(6.4±1.0)%、(6.5±1.3)%(F=139.9,P值均小于0.05),細胞被阻滯在G2/M期.(4)與對照組比較,反義鏈各濃度組存活素蛋白錶達量減少,caspase-3活性明顯增高(F=63.1,P值均小于0.05);正義鏈組、錯義鏈組、脂質體組caspase-3活性與對照組比較,差異無統計學意義(F=0.512,P值均大于0.05).結論 存活素ASODN能夠呈濃度-時間依賴性抑製hMMC株A375增殖,誘導G2/M期阻滯,促進其凋亡.
목적 료해존활소반의과탈양핵감산(ASODN)대인악성흑색소류세포(hMMC)증식화조망적영향.방법 취대수생장기hMMC주A375,안수궤수자표법분위대조조(불전염)、정의련조(전염600 nmol/L존활소정의과탈양핵감산)、착의련조(전염600 nmol/L존활소착의과탈양핵감산)、지질체조(부용지질체처리)、반의련조(전염존활소ASODN,근거전염물농도재분위200、400、600 nmol/L 3개아조),도치형광현미경하관찰전염효과.채용새서람법측정세포존활정황병계산세포증식억제솔,쌍변량류식세포의검측세포조망솔급세포주기,단백질인적법검측존활소단백적표체,격매법검측반광안산천동안산단백매3(caspase-3)적활성.대수거진행방차분석.결과 (1)정의련조、착의련조、반의련600 nmol/L조세포전염솔균대우80%.(2)반의련200、400、600nmol/L조전염후24 h세포증식억제솔[(10.30±0.56)%、(16.69±0.58)%、(24.67±0.67)%]교정의련조[(5.23±0.25)%]、착의련조[(5.09±0.13)%]、지질체조[(4.70±0.45)%]현저증가(F=746.91,P치균소우0.05),차수전염시간연장,증식억제솔증가명현.(3)반의련200、400、600nmol/L조전염후24 h세포조망솔분별위(13.5±1.9)%、(20.1±1.5)%、(32.1±2.9)%,현저고우대조조、정의련조、착의련조、지질체조적(6.5±0.6)%、(5.6±0.7)%、(6.4±1.0)%、(6.5±1.3)%(F=139.9,P치균소우0.05),세포피조체재G2/M기.(4)여대조조비교,반의련각농도조존활소단백표체량감소,caspase-3활성명현증고(F=63.1,P치균소우0.05);정의련조、착의련조、지질체조caspase-3활성여대조조비교,차이무통계학의의(F=0.512,P치균대우0.05).결론 존활소ASODN능구정농도-시간의뢰성억제hMMC주A375증식,유도G2/M기조체,촉진기조망.
Objective To investigate the effect of survivin antisense oligodeoxynucleuotide ( ASODN) on proliferation and apoptosis of human malignant melanoma cells. Methods hMMC A375 colonies in log growth phase were collected and divided into control group (C, without transfection) , sense chain group [ SC, transfected with 600 nmol/L survivin sense oligodeoxynucleuotide ( ODN ) ] , mismatch chain group ( MC, transfected with 600 nmol/L survivin mismatch sense ODN) , liposome group ( L, treated with liposome), antisense chain group (AC, transfected with survivin ASODN, and subdivided into AC 200, 400, 600 nmol/L subgroups) according to the random number table. Transfection result was observed under inverted fluorescence microscope. Inhibition rate of cell proliferation was calculated after determination of cell viability with MTT method. Cell cycle and apoptosis rate were detected with bi-variable flow cytome-try. Expression of survivin protein was determined with Western blot. Activity of caspase-3 was assessed with kinase method. Data were processed with analysis of variance. Results ( 1 ) Cell transfection rates in SC, MC, AC 600 nmol/L groups were all above 80%. (2) Compared with those in SC group [ (5.23 ± 0. 25)% ] , MC group [ (5.09 ±0. 13)% ] and L group [ (4. 70 ±0.45)% ] , inhibition rates of cell proliferation in AC 200, 400, 600 nmol/L groups 24 hours after transfection [ ( 10. 30 ± 0. 56)% , ( 16. 69 ± 0. 58) % , ( 24. 67 ± 0. 67 ) % ] were significantly increased ( F = 746. 91, and P values all below 0. 05 ). As time after transfection went on, proliferation inhibition rate was increased obviously. (3) Apoptosis rate in AC 200, 400, 600 nmol/L groups 24 hours after transfection was respectively (13.5 ±1.9)% , (20. 1 ± 1.5)% , (32. 1 ±2.9)% , which were significantly higher than those in C, SC, MC, and L groups [ (6.5 ± 0.6)%, (5.6 ±0.7)% , (6.4 ± 1.0)% , (6.5±1.3)% ,F = 139.9, P values all below 0.05]. Cells in AC group were blocked in G2/M stage. (4) Compared with those in C group, expression amount of survivin protein decreased, and caspase-3 activity obviously increased ( F =63. 1 , P values all below 0.05) in AC group. No significant difference in caspase-3 activity between SC, MC, L groups and C group was observed ( F =0.512, P values all above 0.05). Conclusions Survivin ASODN can inhibit the proliferation of hMMC A375 in a concentration-time dependant manner, and it induces G2/M stage block and promotes its apoptosis.
