【摘要】：目的 觀察腎損傷分子-1(Kim-1)特異性小干擾RNA（siRNA）沉默Kim-1基因后，對腎細(xì)胞癌細(xì)胞株786-O細(xì)胞的細(xì)胞增殖和細(xì)胞周期的影響。探討Kim-1作為腎細(xì)胞癌靶向治療的新靶點的可能。 方法 采用脂質(zhì)體介導(dǎo)方法將3條對人Kim-1基因序列特異的siRNA序列(50nmol/L)轉(zhuǎn)染進(jìn)入腎細(xì)胞癌786-O細(xì)胞中，轉(zhuǎn)染后24h在熒光顯微鏡下觀察轉(zhuǎn)染效率。轉(zhuǎn)染后48h采用實時定量聚合酶鏈反應(yīng)（qRT-PCR）檢測Kim-1基因的沉默效果，選取沉默效果較好的一組進(jìn)行后續(xù)檢測。采用噻唑藍(lán)（MTT）比色法檢測不同時間點（轉(zhuǎn)然后24h，48h，72h，96h）細(xì)胞增殖的情況，轉(zhuǎn)染后48h采用流式細(xì)胞儀檢測細(xì)胞周期的分布情況。 結(jié)果 1.脂質(zhì)體介導(dǎo)的轉(zhuǎn)染能有效地將siRNA序列轉(zhuǎn)染進(jìn)入786-O細(xì)胞中，轉(zhuǎn)染有效率為（94.36±2.57）%。 2. Kim-1-siRNA能有效下調(diào)786-O細(xì)胞中Kim-1基因的表達(dá)水平，三個不同序列的Kim-1-siRNA的沉默效果分別為（72.47±5.91）%、（12.98±2.21）%、（29.56±3.88）%；而陰性對照組的相對表達(dá)量為（96.56±3.47）%（P0.05）。 3.在腎細(xì)胞癌中沉默Kim-1基因的表達(dá)后，細(xì)胞的增殖受到抑制，3組轉(zhuǎn)染后96h的增殖抑制率分別為空白對照組0%、陰性對照組(3.80±1.24)%、實驗組(54.31±3.82)%（P0.05）。 4.在腎細(xì)胞癌中沉默Kim-1基因的表達(dá)后使細(xì)胞周期阻滯在G0/G1期，轉(zhuǎn)染48h后檢測各組細(xì)胞G0/G1期所占的比例分別為空白對照組（38.44±1.82）%、陰性對照組（42.06±2.15）%、實驗組（63.69±2.87）%（P0.05）。 結(jié)論 1. Kim-1特異性siRNA可有效沉默腎細(xì)胞癌細(xì)胞株786-O中Kim-1的表達(dá)。 2.在腎細(xì)胞癌細(xì)胞株786-O中沉默Kim-1分子的表達(dá)，可使細(xì)胞周期阻滯在G0/G1期，抑制細(xì)胞增殖。 3. Kim-1分子在腎細(xì)胞癌的發(fā)生和發(fā)展中可能具有重要的作用，，可作為腎細(xì)胞癌治療潛在的特異性靶點。
[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 鹵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 鹵5.91)%, (12.98 鹵2.21)%, (29.56 鹵3.88)%, respectively. The relative expression in negative control group was (96.56 鹵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 鹵1.24)% in the negative control group. The experimental group (54.31 鹵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 鹵1.82)%. Negative control group (42.06 鹵2.15)%, experimental group (63.69 鹵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.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R737.11

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