过表达Notchl基因对前列腺癌细胞增殖和周期的影响

Effect of Notchl gene overexpression on proliferation and cell cycle of human prostate cancer cell line PC-3

目的观察Notehl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Notchl—ORF质粒，采用实时定量聚合酶链反应（Real-timePCR）和Westernblot检测Notchl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490nm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Notchl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±0．01，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、72h的4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Notchl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Notchl负性调节Ki-67的表达实现的。

Objective To study the effect of Notchl gene overexpression on proliferation and cell cycle of human prostate cancer cell line PC-3. Methods Notchl-ORF plasmid and its blank vector were transfected into PC-3 cells respectively. The expression of Notehl 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-Dimethyhhiazol-2-yl）-5-（3-carboxymethoxyphenyl）-2-（4-sulfophenyl）-2H-tetrazolium （MTS） assay （20 μl/well） and flow cytometry respectively. Results Compared to negative control （3. 574 ± 0. 368 ） and untransfected group （ 3. 306 ± 0. 278）, Notehl expression in NotcH-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 Notehl-ORF group, negative control group and untransfected group, respectively （P 〈0. 01 ）. MTS assay revealed that the absorbance value in Notchl-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 Notchl-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 Notchl gene in PC-3 cells promoted arrest of G0/G1 phase and suppressed cell proliferation probably by down-regulating Ki-67.