RNA干扰沉默CyclinD1基因对胰腺癌AsPC-1细胞增殖和凋亡的影响

摘要

目的 探讨以细胞周期蛋白(Cyclin) D1为靶基因的RNA干扰对胰腺癌AsPC-1细胞增殖和凋亡的影响,为胰腺癌的靶向治疗提供依据.方法 用含有10％ FBS的DMEM培养液在37℃、5％CO2培养箱中常规培养AsPC-1细胞,至对数生长期后接种于96孔培养板中,随机分为实验组、阴性对照组、空白对照组,并采用LipofectamineTM2000脂质体分别转染Cyclin D1-小干扰RNA(siRNA)、阴性对照siRNA、脂质体,48 h时收集细胞.应用荧光定量PCR法和Western blot法分别检测细胞中Cyclin D1 mRNA和蛋白表达水平,MTT法检测细胞体外增殖活力,流式细胞仪检测细胞周期分布及凋亡率(AI).结果与空白对照组和阴性对照组相比,实验组Cyclin D1 mRNA和蛋白表达均明显下调,细胞生长速度明显减慢,Go/G1细胞比例显著增大、S期细胞比例则明显降低、AI显著升高(P均＜0.01).结论 Cyclin D1-siRNA能通过沉默靶基因表达抑制AsPC-1细胞生长、改变细胞周期分布、诱导细胞凋亡,可作为胰腺癌基因治疗的一个有效靶点.%Objective To investigate the effect of silencing Cyclin Dl gene on the proliferation, cell cycle and apopto-sis of pancreatic carcinoma AsPC-1 cells by RNA interference, and provide evidence for the targeted therapy of pancreatic carcinoma. Methods AsPC-1 cells were cultured in DMEM supplemented with 10% fetal bovine serum, and incubated in a cell incubator with 5% CO2 at 37 ℃. Cells at exponential growth phase were planted on 96 well culture plate, and randomly divided into experimental group, negative control group and blank control group, Cyclin Dl-siRNA, negative-siRNA and lipofectamine were transfected into AsPC-1 cells mediated by Lipofectamine TM 2000, and the cells were collected at 48 h after transfection. The expression of Cyclin Dl Mrna and protein of the transfected cells were analyzed by real-time RT-PCR and Western blot, cell growth was measured with MTT assay, cell cycle and apoptosis rate (AI) was examined by flow cy tome try. Results The expression of Cyclin Dl Mrna and protein in experimental group were markedly down-regulated compared with control groups (P < 0.01). The cell growth of experimental group was significantly slower than that of control groups (P <0.01). The percentage of G0/G1 stage cells was significantly higher, while the percentage of S stage cells was significantly lower in experimental group compared with those in control groups (P <0.01). The apoptosis rate of experimental group was significantly higher than that of control groups (P <0.01). Conclusion Down-regulation of Cyclin Dl can inhibit the growth of pancreatic carcinoma AsPC-1 cells, change cell cycle distribution and induce cell apoptosis. Cyclin Dl gene may serve as an effective target for the gene therapy of pancreatic carcinoma.