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siRNA沉默NGAL基因对结肠癌细胞行为的影响
作者:王厚东  沈忠  杨关根  鲁振锋  陈金明  刘道成  李良飞 
单位:安徽医科大学杭州临床学院  杭州市第三人民医院肛肠外科  浙江 杭州 310009 
关键词:结肠肿瘤/病理学 RNA 小分子干扰 癌基因蛋白质类/代谢 急相蛋白质类/代谢 明胶酶类/代谢 中性白细胞/酶学 基因沉默 细胞增殖 细胞运动 细胞凋亡 
DOI:R735.3+5;R730.23
出版年,卷(期):页码:2018,33(2):122-127
摘要:

目的 探讨中性粒细胞明胶酶相关载脂蛋白(neutrophils gelatinases-associated lipocalin,NGAL)的基因沉默作用对结肠癌细胞增殖、迁移和凋亡的影响。方法 采用转染试剂盒ribo FECTTM CP Transfection Kit负载NGAL siRNA转染结肠癌细胞株HT-29(转染组),以未转染的HT-29细胞为对照组。实时荧光定量PCR和ELISA分别检测细胞NGAL mRNA水平和细胞培养上清液中NGAL蛋白水平,采用细胞划痕实验、Promega Cell Proliferation Assay G3580和BD C6流式细胞仪分别检测细胞迁移、增殖和凋亡情况。结果 HT-29细胞转染NGAL siRNA 72 h后,转染组NGAL mRNA水平为对照组的0.123(P<0.05);转染48 h后,转染组细胞培养上清液中NGAL蛋白为(0.328±0.055)μg/L,低于对照组的(1.071±0.143)μg/L (t=8.205,P=0.001)。转染48 h后,转染组凋亡率为(18.87±1.65)%,低于对照组的(11.93±1.90)%(t=-4.752,P=0.009)。转染24 h后,转染组细胞迁移率为对照组的(23.35±6.35)%(t=11.930,P<0.01)。转染48 h后,转染组细胞增殖率为对照组的(90.00±5.30)%(t=1.983,P=0.118)。结论 NGAL siRNA可以抑制结肠癌细胞NGAL基因和蛋白的表达,抑制细胞迁移,促进细胞凋亡。

Objective To explore the impact of neutrophils gelatinases-associated lipocalin (NGAL) gene silencing on the proliferation, migration and apoptosis of colon cancer cells. Methods The colon cancer cell line HT-29 was transfected with NGAL siRNA, which was encapsulated by ribo FECTTM CP Transfection Kit (TK), and set as the transfection group. The untransfected(empty vector transfected) HT-29 cells were set as the control group. The levels of NGAL mRNA and protein were detected by real-time quantitative PCR (RT-QPCR) and enzyme linked immunosorbent assay (ELISA) respectively. Cell migration, proliferation and apoptosis were examined by w365bet娱乐官网网址ound healing assay, Promega Cell Proliferation Assay G3580 and BD C6 flow cytometry. Results At 72 h after NGAL siRNA transfection, the level of NGAL mRNA in the transfection group was 0.123 of the control group, which was set as 1.000 (P<0.05). At 48 h after transfection, the level of NGAL protein in the cell culture supernatant of the transfection group was significantly lower than that of the control group[(0.328±0.055)μg/L vs (1.071±0.143)μg/L, t=8.205, P=0.001]. At the same time point, the apoptosis rate was also significantly reduced in the transfection group compared with the control group[(18.87±1.65)% vs (11.93±1.90)%, t=-4.752, P=0.009]. At 24 h after transfection, the migration rate of the transfection group was decreased to only (23.35±6.35)% of the control group (t=11.930, P<0.01). Moreover, the cell proliferation rate of the transfection group was (90.00±5.30)% of the control group (t=1.983, P=0.118) at 48 h after transfection. Conclusion NGAL siRNA can inhibit the expression of NGAL in colon cancer cells, which subsequently inhibit cell migration and enhance cell apoptosis.

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