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背景与目的: 肝细胞癌是严重威胁人类健康的恶性肿瘤之一, p53又是重要的抑癌基因,而RNA干扰(RNAinterference, RNAi) 不仅是一种经济、快捷、高效的抑制基因表达的技术手段, 而且有可能在肿瘤基因治疗方面提供新的途径。因此, 我们将外源性p53双链小RNA(smalldoublestrandedRNA, dsRNA) 瞬时转染肝癌SK- Hep -1细胞系, 观察其对肝癌细胞P53和P21蛋白表达及细胞周期的影响。方法: 合成的p53dsRNA和EGFPdsRNA, 分别以200ng和400ng的p53dsRNA用脂质体转染法转染SK- Hep- 1细胞, 同时设0. 9%NaCl空白对照和EG- FP及EGFP+EGFPdsRNA阳性对照组, 每组3个复孔, 分别在转染24h和48h时用流式细胞仪进行细胞周期检测, 对转染p53dsRNA48h后应用WesternBlot检测P53和P21蛋白的表达,初步探讨p53的dsRNA干扰对肝癌细胞影响的机制。结果: SK- Hep -1细胞在转染200ngp53dsRNA后G0 -G1 期细胞明显减少, 24h时与空白对照相比减少了52 .53%, 与转染同剂量EG FP+EGFPdsRNA的阳性对照组相比也减少了50 .29% (P<0. 05); S期细胞明显增多, 24h时与空白对照相比增加了146 8%, 与转染同剂量EGFP+EGFPdsRNA的阳性对照组相比增加了128. 62% (P<0 .05); G2-M期细胞也明显增多, 24h时与空白对照相比增加了30. 56% (P<0. 05),
BACKGROUND & OBJECTIVE: Hepatocellular carcinoma (HCC) is one of the most malignant tumors that threaten human health. P53 is an important tumor suppressor gene. RNA interference (RNAi) is not only an economical, rapid and efficient technique to inhibit gene expression Means, and may provide new ways in the gene therapy of cancer. Therefore, we transiently transfected small double double-stranded RNA (dsRNA) into SK-Hep-1 cells and observed the effect on the expression and cell cycle of P53 and P21. Methods: The synthesized p53dsRNA and EGFP dsRNA were transfected into SK-Hep-1 cells with 200ng and 400ng of p53dsRNA by lipofectamine 2000 respectively. At the same time, 0.9% NaCl control, EG-FP and EGFP + EGFP dsRNA positive control Group, with 3 replicate wells in each group. The cell cycle was detected by flow cytometry at 24h and 48h respectively. The expression of P53 and P21 protein was detected by Western Blot 48 hours after transfected with p53dsRNA, and the preliminary study of p53 dsRNA interference on liver cancer Mechanism of cell effects. Results: After transfected with 200 ngp53 dsRNA, the number of SK-Hep-1 cells decreased significantly at G0-G1 phase and decreased by 52.53% at 24 hours compared with the positive control group transfected with the same dose of EG FP + EGFP dsRNA Also decreased by 50.29% (P <0.05). The number of S phase cells increased significantly at 24h, increased by 146.8% compared with the blank control group, increased compared with the positive control group transfected with the same dose of EGFP + EGFPdsRNA 128. 62% (P <0 .05); G2-M phase cells also increased significantly, compared with the blank control 24h increased by 30.56% (P <0.05)