中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2013年
1期
20-22
,共3页
冷政伟%夏清华%殷涛%陶凯雄
冷政偉%夏清華%慇濤%陶凱雄
랭정위%하청화%은도%도개웅
胰腺癌%Bmi-1%甲基化%p16
胰腺癌%Bmi-1%甲基化%p16
이선암%Bmi-1%갑기화%p16
Pancreatic carcinoma%Bmi-1%Methylation%p16
目的 观察靶向下调Bmi-1基因对人胰腺癌细胞(PANC-1)细胞增殖、p16启动子甲基化、p16表达的影响.方法 构建反义Bmi-1真核表达载体转染人人胰腺癌PANC-1细胞,运用荧光倒置显微镜观察、Western blot技术检测靶基因沉默效率；运用流式细胞术检测细胞周期及凋亡；Western blot、实时定量聚合酶链反应(Real-time PCR)检测p16基因表达；甲基化特异性聚合酶链反应(MSP)检测p16启动子甲基化变化.结果 荧光倒置显微镜观察转染后48 h细胞转染效率在90％左右；细胞周期出现阻滞(G0/G1期细胞由40.52％上升至60.48％,P＜0.05)、凋亡增加(由7.87％上升至21.67％,P＜0.05)；Bmi-1蛋白表达水平明显下降[吸光度(IA)值318.54±1.21到175.39±0.73,P＜0.05]；p16启动子甲基化水平降低而p16基因RNA(从3.563±0.128上升到8.621±0.310,P＜0.05)及蛋白质表达上升(IA值213.38±0.54到304.12±0.76,P＜0.05).结论 本实验构建的反义Bmi-1载体能够有效沉默细胞中靶基因的表达,抑制细胞增殖；Bmi-1很可能通过甲基化的方式对p16的表达进行调控.
目的 觀察靶嚮下調Bmi-1基因對人胰腺癌細胞(PANC-1)細胞增殖、p16啟動子甲基化、p16錶達的影響.方法 構建反義Bmi-1真覈錶達載體轉染人人胰腺癌PANC-1細胞,運用熒光倒置顯微鏡觀察、Western blot技術檢測靶基因沉默效率；運用流式細胞術檢測細胞週期及凋亡；Western blot、實時定量聚閤酶鏈反應(Real-time PCR)檢測p16基因錶達；甲基化特異性聚閤酶鏈反應(MSP)檢測p16啟動子甲基化變化.結果 熒光倒置顯微鏡觀察轉染後48 h細胞轉染效率在90％左右；細胞週期齣現阻滯(G0/G1期細胞由40.52％上升至60.48％,P＜0.05)、凋亡增加(由7.87％上升至21.67％,P＜0.05)；Bmi-1蛋白錶達水平明顯下降[吸光度(IA)值318.54±1.21到175.39±0.73,P＜0.05]；p16啟動子甲基化水平降低而p16基因RNA(從3.563±0.128上升到8.621±0.310,P＜0.05)及蛋白質錶達上升(IA值213.38±0.54到304.12±0.76,P＜0.05).結論 本實驗構建的反義Bmi-1載體能夠有效沉默細胞中靶基因的錶達,抑製細胞增殖；Bmi-1很可能通過甲基化的方式對p16的錶達進行調控.
목적 관찰파향하조Bmi-1기인대인이선암세포(PANC-1)세포증식、p16계동자갑기화、p16표체적영향.방법 구건반의Bmi-1진핵표체재체전염인인이선암PANC-1세포,운용형광도치현미경관찰、Western blot기술검측파기인침묵효솔；운용류식세포술검측세포주기급조망；Western blot、실시정량취합매련반응(Real-time PCR)검측p16기인표체；갑기화특이성취합매련반응(MSP)검측p16계동자갑기화변화.결과 형광도치현미경관찰전염후48 h세포전염효솔재90％좌우；세포주기출현조체(G0/G1기세포유40.52％상승지60.48％,P＜0.05)、조망증가(유7.87％상승지21.67％,P＜0.05)；Bmi-1단백표체수평명현하강[흡광도(IA)치318.54±1.21도175.39±0.73,P＜0.05]；p16계동자갑기화수평강저이p16기인RNA(종3.563±0.128상승도8.621±0.310,P＜0.05)급단백질표체상승(IA치213.38±0.54도304.12±0.76,P＜0.05).결론 본실험구건적반의Bmi-1재체능구유효침묵세포중파기인적표체,억제세포증식；Bmi-1흔가능통과갑기화적방식대p16적표체진행조공.
Objective To study the effect of knocking down B-cell specific moloney leukemia virus insertion site 1 (Bmi-1) on cell proliferation,the methylation of p16 and the expression of p16.Methods Antisense Bmi-1 vector was constructed and transfected into human pancreatic cancer cell line (PANC-1) cells,and the efficiency of transfection was evaluated by fluorescence microscope and Western blotting.Cell cycle and apoptosis were examined by using flow cytometry.The expression of p16 and the methylation of p16 promotor were detected by using Western blotting,real-time polymerase chain reaction (PCR) and methylation-specific PCR,respectively.Results Antisense Bmi-1 vector was successfully constructed,and the transfection efficiency was about 90％.The percentage of G0G1 cells was increased from 40.52％ to 60.48％ (P ＜ 0.05) and apoptosis rate increased from 7.87％ to 21.67％ (P ＜ 0.05).The expression of Bmi-1 protein was reduced from 318.54 ± 1.21 to 175.39 ±0.73 (P ＜0.05),the expression levels of p16 RNA and protein in the transfected cells were increased to 8.621 ± 0.310 and 304.12 ±0.76 respectively (P ＜ 0.05),and the promotor methylation of p16 was declined (P ＜ 0.05).Conclusion Antisense Bmi-1 vector may knock down the expression of Bmi-1 in PANC-1 cells and could inhibit the proliferation of cells.The expression of p16 is possibly regulated by Bmi-1 via promoting the methvlation of o16 oromotor.
