过表达Numb基因对人膀胱癌5637细胞周期和增殖的影响

目的：：探讨 Numb 基因对人膀胱癌细胞周期和增殖能力的影响及相关机制。方法：选取人膀胱癌细胞5637为研究对象,采用 Numb-ORF 表达质粒转染细胞为实验组,设置阴性对照和空白对照组。采用荧光定量聚合酶链式反应和蛋白质印迹技术检测各组中 Numb 基因和细胞周期蛋白 D1（Cyclin D1）的表达;采用流式细胞技术检测各组的细胞周期;采用酶标仪检测细胞在490nm 的吸光度值,评价各组细胞的增殖活力。结果：实验组 Numb的△CT 值为1.58±0.41,阴性对照组为5.66±0.53,空白对照组为5.81±0.47,差异有统计学意义（F ＝77.39, P ＝0.00）;实验组 Cyclin D1的△CT 值为4.92±0.73,阴性对照组为2.59±0.33,空白对照组为2.45±0.26,差异有统计学意义（F ＝11.70,P ＝0.01）。实验组中 G0／G1期细胞的比例（％）为49.76±7.07,明显高于阴性对照组的36.52±3.32和空白对照组的38.21±2.06,差异有统计学意义（F ＝7.17,P ＝0.03）。增殖实验中,实验组24 h 的吸光度值为0.35±0.08,明显低于阴性对照组0.52±0.06和空白对照组0.55±0.04（F ＝9.03,P ＝0.02）。结论：Numb 基因可以提高 G0／G1期细胞比例,抑制膀胱癌细胞增殖,其机制可能是通过抑制 Cyclin D1表达实现。

Effect of Numb Gene on Cell Cycle and Proliferation in Human Bladder Cancer 5637 Cells

Objective:To study the effect of Numb gene on cell cycle and proliferation in human bladder cancer cells and its related mechanism.Methods:Human bladder cancer 5637 cells which were transfected by the Numb-ORF plasmid were used as the experimental group.The negative control and blank control were also set.The expression of Numb or Cyclin D1 was detected by Real-Time PCR and Western Blot.The cell cycle was analyzed by Flow cytometry.Cell pro-liferation was assessed by comparing with absorbance at 490nm using a Micro-plate Reader.Results:Compared with the negative control (5.66 ±0.53)and blank control (5.81 ±0.47),the △CT value of Numb in the Numb-ORF group (1.58 ±0.41)was significantly lower (F =77.39,P =0.00).Meanwhile,the △CT value of Cyclin D1 in the Numb-ORF group (4.92 ±0.73)was significantly higher than that in the negative control (2.59 ±0.33)and blank control (2.45 ± 0.26)(F =11.70,P =0.01).The ratio (%)of G0 /G1 cells was as follows:Numb-ORF group was 49.76 ±7.07,nega-tive control was 36.52 ±3.32 and blank control was 38.21 ±2.06 (F =7.17,P =0.03).In proliferation assay,compared with negative control (0.52 ±0.06)and blank control (0.55 ±0.04),the OD value at 24h in Numb-ORF group (0.35 ± 0.08)was significantly reduced (F =9.03,P =0.02).Conclusion:Numb gene could promote G0 /G1 phase arrested and inhibit proliferation of bladder cancer cells,the suppressive effects may be due to down-regulation of Cyclin D1.