CAJ | 학술논문

目的观察Notch1基因对前列腺癌细胞增殖和周期的影响.方法在前列腺癌细胞PC-3中转染Notch1-ORF质粒,采用实时定量聚合酶链反应(Real-time PCR)和Western blot检测Notch1和细胞核增殖抗原(Ki-67)的表达.以四唑氮化合物(MTS)试剂(每孔20μl)加入细胞,检测490 nm处吸光度(A)值评价细胞增殖能力.以碘化丙锭反应液1ml染色细胞,流式细胞仪检测细胞周期的变化.结果实验组Notch1基因表达水平为43.740±6.438,明显高于阴性对照组(3.574±0.368)和空白对照组(3.306±0.278);同时实验组Ki-67的表达减低,实验组为0.576±0.063,阴性对照组为1.012±0.011,空白对照组为1.000±0.007 (P＜0.01).MTS实验中实验组细胞在24、48、72 h的A值均低于两对照组细胞(P＜0.01).实验组G0/G1期比例为(64.013±1.952)％,明显高于阴性对照组的(48.917 ±2.771)％和空白对照组的(51.317±1.907)％,差异有统计学意义(P＜0.01).结论前列腺癌细胞中Notch1的表达可以促使细胞发生G0/G1期阻滞,抑制细胞增殖能力,这种抑制作用可能是通过Notch1负性调节Ki-67的表达实现的.
목적 관찰Notch1기인대전렬선암세포증식화주기적영향.방법 재전렬선암세포PC-3중전염Notch1-ORF질립,채용실시정량취합매련반응(Real-time PCR)화Western blot검측Notch1화세포핵증식항원(Ki-67)적표체.이사서담화합물(MTS)시제(매공20μl)가입세포,검측490 nm처흡광도(A)치평개세포증식능력.이전화병정반응액1ml염색세포,류식세포의검측세포주기적변화.결과 실험조Notch1기인표체수평위43.740±6.438,명현고우음성대조조(3.574±0.368)화공백대조조(3.306±0.278);동시실험조Ki-67적표체감저,실험조위0.576±0.063,음성대조조위1.012±0.011,공백대조조위1.000±0.007 (P＜0.01).MTS실험중실험조세포재24、48、72 h적A치균저우량대조조세포(P＜0.01).실험조G0/G1기비례위(64.013±1.952)％,명현고우음성대조조적(48.917 ±2.771)％화공백대조조적(51.317±1.907)％,차이유통계학의의(P＜0.01).결론 전렬선암세포중Notch1적표체가이촉사세포발생G0/G1기조체,억제세포증식능력,저충억제작용가능시통과Notch1부성조절Ki-67적표체실현적.
Objective To study the effect of Notch1 gene overexpression on proliferation and cell cycle of human prostate cancer cell line PC-3.Methods Notch1-ORF plasmid and its blank vector were transfected into PC-3 cells respectively.The expression of Notch1 or Ki-67 was detected by real-time quantitative polymerase chain reaction (Real-time PCR) and Western blotting.The cell proliferation and cell cycle were analyzed by 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium (MTS) assay (20 μ l/well) and flow cytometry respectively.Results Compared to negative control (3.574 ±0.368) and untransfected group (3.306 ± 0.278),Notch1 expression in Notch1-ORF group (43.740 ± 6.438) was significantly increased (P ＜ 0.01).Meanwhile,proliferating cell nuclear antigen (Ki-67) level was decreased:0.576 ± 0.063,1.012 ± 0.011,and 1.000 ± 0.007 in Notch1-ORF group,negative control group and untransfected group,respectively (P ＜0.01).MTS assay revealed that the absorbance value in Notch1-ORF group was significantly reduced at 24,48 and 72 h (P ＜ 0.01).Flow cytometry analysis showed that the proportion of cells in G0/G1 phase in Notch1-ORF group was (64.013 ± 1.952) ％,significantly higher than in negative control group [(48.917 ± 2.771) ％] and blank control group [(51.317 ± 1.907)％] (P＜0.01).Conclusion Overexpression of Notch1 gene in PC-3 cells promoted arrest of G0/G1 phase and suppressed cell proliferation probably by down-regulating Ki-67.