摘要
目的探讨小干扰RNA（siRNA）沉默表皮生长因子受体（EGFR）对血管平滑肌细胞（VSMC）增殖和迁移的影响及其机制。方法分离、培养大鼠VSMC，取对数生长期细胞，利用慢病毒载体感染VSMC，将细胞分为siRNA-EGFR组、siRNA-阴性对照组和空白对照组，5-溴脱氧尿嘧啶核苷（5-BrdU）法检测各转染组细胞增殖能力，Transwell小室和划痕实验检测各转染组细胞迁移能力，实时定量聚合酶链反应（Real-time PCR）检测各转染组细胞中EGFR、细胞外信号调节激酶（ERK）、c-jun氨基末端激酶（JNK）、p38丝裂原活化蛋白激酶（p38）基因表达，Western blot法检测各转染组细胞中EGFR、磷酸化ERK（p-ERK）、磷酸化JNK（p-JNK）和磷酸化p38（p-p38）蛋白表达。结果siRNA-EGFR组VSMC迁移细胞数[（48.5±6.1）个]，均显著低于siRNA-阴性对照组和空白对照组，分别为（86.2±7.2）个和（88.3±7.6）个，差异均有统计学意义（F=37.261，P=0.000），与0 h比较，24 h后，siRNA-EGFR组VSMC迁移率为（35.7±3.8）%，显著低于siRNA-阴性对照组和空白对照组，分别为（74.2±5.7）%和（75.5±6.1）%，差异均有统计学意义（F=40.315，P=0.000）；siRNA-EGFR组细胞中ERK、JNK和p38 mRNA相对表达量（0.53±0.09、0.47±0.11、0.41±0.07）均低于siRNA-阴性对照组（0.82±0.12、0.64±0.10、0.68±0.09）和空白对照组（0.85±0.14、0.66±0.13、0.70±0.12），差异均有统计学意义（F=15.391、12.817、11.264，P=0.000、0.000、0.000）；siRNA-EGFR组细胞中p-ERK、p-JNK和p-p38蛋白相对表达量（0.46±0.09、0.38±0.06、0.33±0.06）均低于siRNA-阴性对照组（0.71±0.12、0.57±0.08、0.52±0.07）和空白对照组（0.73±0.13、0.56±0.07、0.54±0.09），差异均有统计学意义（F=20.152、14.375、13.843，P=0.000、0.000、0.000）。结论特异性抑制EGFR可有效减少VSMC增殖和迁移能力，其机制可能与抑制丝裂原活化蛋白激酶（MAPK）信号通路有关。
ObjectiveTo investigate the effect of silencing epidermal growth factor receptor （EGFR） by small interfering RNA （siRNA） interference on the proliferation and migration of vascular smooth muscle cells （VSMCs）, and the related mechanisms.MethodsVSMCs of rats were isolated and cultured. VSMCs were infected with EGFR siRNA lentiviral vectors. The cells were divided into siRNA-EGFR group, siRNA-negative control group and blank control group. The cell proliferation abilities were detected by 5’-bromo-2’-deoxyuridine （5-BrdU） method. The cell migration abilities in different transfection groups were examined by Transwell chamber and scratch assays. The expression levels of EGFR, extracellular signal-regulated kinase （ERK）, c-Jun N-terminal kinase （JNK） and p38 genes in different transfection groups were detected using real-time quantitative polymerase chain reaction （Real-time PCR） technology. The expression levels of EGFR, p-ERK, p-JNK and p-p38 proteins in different transfection groups were detected by Western blotting.ResultsThe number of migration cells in the siRNA-EGFR group was 48.5±6.1, which were significantly lower than the siRNA-negative control group and blank control group, which were 86.2±7.2 and 88.3±7.6; the difference was statistically significant （F=37.261, P=0.000）. Compared with 0 h, after 24 h, the migration rate of VSMC in the siRNA-EGFR group was （35.7±3.8）%, which was significantly lower than that of siRNA-EGFR group and blank group, which were （74.2±5.7）% and （75.5±6.1）%; the differences were statistically significant （F=40.315, P=0.000）. The relative expression levels of ERK, JNK and p38 mRNA in the siRNA-EGFR group （0.53±0.09, 0.47±0.11, 0.41±0.07） were lower than the siRNA-negative control group （0.82±0.12, 0.64±0.10, 0.68±0.09） and blank control group （0.85±0.14, 0.66±0.13, 0.70±0.12）, the differences were statistically significant （F=15.391, 12.817, 11.264, P=0.000, 0.000, 0.000）. The relative expression levels of p-ERK, p-JNK and p-p38 proteins in the siRNA-EGFR group （0.46±0.09, 0.38±0.06, 0.33±0.06） were lower than the siRNA-negative control group （0.71±0.12, 0.57±0.08, 0.52±0.07） and blank control group （0.73±0.13, 0.56±0.07, 0.54±0.09）, the differences were statistically significant （F=20.152, 14.375, 13.843, P=0.000, 0.000, 0.000）.ConclusionSpecific inhibition of EGFR could effectively reduce proliferation and migration capacities of VSMCs, which might be related to inhibition of mitogen-activated protein kinase signaling pathway.
出处
《中华实验外科杂志》
CSCD
北大核心
2017年第5期729-732,共4页
Chinese Journal of Experimental Surgery
基金
2016年河南省科技攻关项目（162102310034）
关键词
血管平滑肌细胞
表皮生长因子受体
小分子干扰
细胞增殖
细胞迁移
Vascular smooth muscle cells
Epidermal growth factor receptor
Small interfer-ence
Cell proliferation
Cell migration
