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目的探讨胰岛素诱导人肾小球系膜细胞(HMC)血管内皮细胞生长因子(VEGF)基因转录机制。方法 (1)用VEGF报告质粒或低氧诱导因子1(HIF-1)结合位点畸变的VEGF mut报告质粒转染HMC,荧光素酶分析法检测其转录活性;(2)应用Real-time PCR、Western blot法分别检测HIF-1α mRNA和蛋白表达。结果 (1)胰岛素呈剂量依赖性增加VEGF基因转录,在100nmol/L时达高峰,为未刺激组的2.14±0.17倍(P<0.01),但对转染VEGF mut报告质粒的HMC VEGF基因转录无影响;(2)Ly294002和雷帕霉素均可抑制胰岛素诱导的VEGF基因转录(P<0.01);(3)胰岛素呈时间依赖性上调HIF-1α蛋白表达,4h达高峰,为对照组的2.35±0.35倍(P<0.01),但对其mRNA表达无影响。结论胰岛素通过激活PI3K/mTOR通路和上调HIF-1α蛋白表达诱导HMC VEGF基因转录。
Objective To investigate the transcriptional mechanism of insulin-induced vascular endothelial growth factor (VEGF) gene in human glomerular mesangial cells (HMC). Methods (1) VEGF mut reporter plasmids with HIF-1 or HIF-1 binding sites were transfected into HMC and luciferase assay was used to detect the transcriptional activity. (2) Real-time PCR The expression of HIF-1α mRNA and protein were detected by Western blot. Results (1) Insulin increased VEGF gene transcription in a dose-dependent manner, reaching a peak at 100 nmol / L, which was 2.14 ± 0.17 times more than that of unstimulated group (P <0.01) (2) Both Ly294002 and rapamycin inhibited insulin-induced VEGF gene transcription (P <0.01). (3) Insulin increased HIF-1α protein expression in a time-dependent manner and peaked at 4h 2.35 ± 0.35 times (P <0.01), but had no effect on its mRNA expression. Conclusion Insulin induces HMC VEGF gene transcription by activating PI3K / mTOR pathway and up-regulating HIF-1α protein expression.