摘要
目的研究Numb基因对人肾癌细胞的细胞周期和增殖能力的影响及相关机制。方法选取人肾癌Caki-1细胞为研究对象，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反应（RT—qPCR）和蛋白质印迹法检测各组中Numb和细胞周期蛋白D1（cvclin D1）的表达，采用流式细胞术检测各组的细胞周期，采用酶标仪检测细胞在波长490nm处的吸光度值，评价各组细胞的增殖活力。结果实验组PCR扩增Numb的△Ct值为1．92±0．39，阴性对照组为5．05±0．45，空白对照组为5．13±0．31；实验组蛋白质相对表达强度为6．67±0．83，阴性对照组为3．08±0．47，空白对照组为2．85±0．36，差异有统计学意义（P=0．00）；实验组cyclin D1的△Ct值为6．20±0．87，阴性对照组为4．35±0．51，空白对照组为4．56±0．31，差异有统计学意义（P=0．02）。实验组蛋白质相对表达强度为5．85±0．72，阴性对照组为10．04．±0．83，空白对照组为11．88±1．26，差异有统计学意义（P=0．00）。实验组中G0／G1期细胞的比例为（54．29±4．15）％，高于阴性对照组的（38．69±2．60）％和空白对照组的（41．28±1．29）％，差异有统计学意义（P=0．00）。增殖实验中，实验组24h的吸光度值为0．67±0．07，低于阴性对照组（0．93±0．10）和空白对照组（1．02±0．06），差异有统计学意义（P=0．00）。结论Numb基因可以提高肾癌Caki-1细胞岛／G。期细胞比例，抑制细胞增殖，其机制可能是通过抑制cyclin D1表达实现的。
Objective To study the effect of Numb gene on cell cycle and proliferation in human renal carcinoma cells and its related mechanism. Methods Renal carcinoma cells Caki-1 were taken as research objects, and the Numb-ORF plasmid transfeeted cells, negative control group and blank control group were set respectively. The expression levels of Numb and eyelin D1 were detected by real-time PCR and Western blot. The cell cycle was analyzed respectively by flow cytometry. Cell proliferation was assessed by comparing with absorbance at 490 nm using a micro-plate reader. Results Compared to the negative control group （5.05±0.45） and blank control group （5.13±0.31）, the △Ct value of Numb in the Numb-ORF group （1.92±0.39） was significantly lower. The protein levels in the Numb-ORF group, the negative control group and the blank control group were 6.67±0.83, 3.08±0.47, 2.85±0.36, respectively （P = 0.00）. Meanwhile, the △Ct value of eyclin D1 in the Numb-ORF group （6.20±0.87） was higher than that in the negative control group （4.35±0.51） and the blank control group （4.56±0.31） （P = 0.02）, and the protein level in the Numb-ORF group （5.85±0.72） was lower than that in the negative control group （10.04±0.83） and the blank control group （11.88±1.26） （P = 0.00）. The ratio of Go/G1 cells in the Numb-ORF group was （54.29±4.15） %, the negative control group was （38.69±2.60） % and the blank control group was （41.28±1.29） % （P = 0.00）. In proliferation assay, compared with the negative control group （0.93±0.10） and the blank control group （1.02±0.06）, the A value at 24 h in the Numb-ORF group （0.67±0.07） was significantly reduced （P = 0.00）. Conclusion Numb gene could increase the cell percentage in Go/Gl phase and inhibit proliferation of renal carcinoma cells via down-regulating the expression of eyclin D1.
出处
《肿瘤研究与临床》
CAS
2016年第1期1-5,共5页
Cancer Research and Clinic
基金
国家自然科学基金（81100561）
