Kim-1特异性siRNA对肾癌细胞株786-0细胞增殖及细胞周期的影响

发布时间：2018-11-18 11:12

【摘要】：目的观察肾损伤分子-1(Kim-1)特异性小干扰RNA（siRNA）沉默Kim-1基因后，对肾细胞癌细胞株786-O细胞的细胞增殖和细胞周期的影响。探讨Kim-1作为肾细胞癌靶向治疗的新靶点的可能。方法采用脂质体介导方法将3条对人Kim-1基因序列特异的siRNA序列(50nmol/L)转染进入肾细胞癌786-O细胞中，转染后24h在荧光显微镜下观察转染效率。转染后48h采用实时定量聚合酶链反应（qRT-PCR）检测Kim-1基因的沉默效果，选取沉默效果较好的一组进行后续检测。采用噻唑蓝（MTT）比色法检测不同时间点（转然后24h，48h，72h，96h）细胞增殖的情况，转染后48h采用流式细胞仪检测细胞周期的分布情况。结果 1.脂质体介导的转染能有效地将siRNA序列转染进入786-O细胞中，转染有效率为（94.36±2.57）%。 2. Kim-1-siRNA能有效下调786-O细胞中Kim-1基因的表达水平，三个不同序列的Kim-1-siRNA的沉默效果分别为（72.47±5.91）%、（12.98±2.21）%、（29.56±3.88）%；而阴性对照组的相对表达量为（96.56±3.47）%（P0.05）。 3.在肾细胞癌中沉默Kim-1基因的表达后，细胞的增殖受到抑制，3组转染后96h的增殖抑制率分别为空白对照组0%、阴性对照组(3.80±1.24)%、实验组(54.31±3.82)%（P0.05）。 4.在肾细胞癌中沉默Kim-1基因的表达后使细胞周期阻滞在G0/G1期，转染48h后检测各组细胞G0/G1期所占的比例分别为空白对照组（38.44±1.82）%、阴性对照组（42.06±2.15）%、实验组（63.69±2.87）%（P0.05）。结论 1. Kim-1特异性siRNA可有效沉默肾细胞癌细胞株786-O中Kim-1的表达。 2.在肾细胞癌细胞株786-O中沉默Kim-1分子的表达，可使细胞周期阻滞在G0/G1期，抑制细胞增殖。 3. Kim-1分子在肾细胞癌的发生和发展中可能具有重要的作用，，可作为肾细胞癌治疗潜在的特异性靶点。
[Abstract]:Objective to investigate the effects of small interference of renal injury molecule-1 (Kim-1) on the proliferation and cell cycle of renal cell carcinoma cell line 786-O after RNA (siRNA) silencing Kim-1 gene. To explore the possibility of Kim-1 as a new target for the targeted therapy of renal cell carcinoma. Methods three siRNA sequences (50nmol/L) specific to human Kim-1 gene were transfected into renal cell carcinoma 786-O cells by liposome mediated transfection. The transfection efficiency was observed under fluorescence microscope 24 hours after transfection. The silencing effect of Kim-1 gene was detected by real-time quantitative polymerase chain reaction (qRT-PCR) 48 hours after transfection. Cell proliferation was detected by thiazolyl blue (MTT) colorimetry at different time points (then 24 h, 48 h, 72 h, 96 h). Flow cytometry was used to detect the distribution of cell cycle at 48 h after transfection. Result 1. Liposome-mediated transfection could effectively transfect the siRNA sequence into 786-O cells, and the transfection efficiency was (94.36 卤2.57)%. 2. Kim-1-siRNA could effectively down-regulate the expression of Kim-1 gene in 786-O cells. The silencing effect of Kim-1-siRNA with three different sequences was (72.47 卤5.91)%, (12.98 卤2.21)%, (29.56 卤3.88)%, respectively. The relative expression in negative control group was (96.56 卤3.47)% (P0.05). 3. After silencing the expression of Kim-1 gene in renal cell carcinoma, the cell proliferation was inhibited. The inhibition rate of proliferation in the three groups at 96 h after transfection was 0 in the blank control group and (3.80 卤1.24)% in the negative control group. The experimental group (54.31 卤3.82)% (P0.05). 4. After silencing the expression of Kim-1 gene in renal cell carcinoma, the cell cycle was blocked in G0/G1 phase. After 48 hours of transfection, the proportion of G0/G1 phase in each group was (38.44 卤1.82)%. Negative control group (42.06 卤2.15)%, experimental group (63.69 卤2.87)% (P0.05). Conclusion 1. Kim-1 specific siRNA could effectively inhibit the expression of Kim-1 in renal cell carcinoma cell line 786-O. 2. Silencing the expression of Kim-1 in renal cell carcinoma cell line 786-O could block cell cycle in G0/G1 phase and inhibit cell proliferation. 3. Kim-1 may play an important role in the genesis and development of renal cell carcinoma and may be a potential specific target for the treatment of renal cell carcinoma.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.11

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