氧化型低密度脂蛋白对血管平滑肌细胞磷酸酶PTEN活性的影响

The Effect of Oxidized Low Density Lipoprotein on PTEN Phosphatase Activity in Rabbit Vascular Smooth Muscle Cells

摘要:

为探讨氧化型低密度脂蛋白促血管平滑肌细胞增殖的细胞内信号转导机制,将兔血管平滑肌细胞分为对照组、低密度脂蛋白组和氧化型低密度脂蛋白组。以细胞计数和噻唑蓝比色法测定细胞增殖能力,Westernblot定量与细胞骨架蛋白同源在10号染色体有缺失的磷酸酶(PTEN)表达水平,免疫沉淀和特异底物diC16PIP3脱磷酸绿色试剂法检测该磷酸酶活性。实验发现氧化型低密度脂蛋白(50mgL)可使细胞计数及噻唑蓝比色吸光值分别增加1.78和3.21倍,而低密度脂蛋白(50mgL)无明显作用。各浓度低密度脂蛋白及氧化型低密度脂蛋白对该磷酸酶蛋白表达水平均无显著影响。氧化型低密度脂蛋白对PTEN活性的抑制在20～50mgL范围内呈浓度依赖性。时间曲线表现为10min作用最强,并可持续达24h(与对照组比较,均P<0.01)。结果提示,氧化型低密度脂蛋白可能通过对负性调节蛋白PTEN磷酸酶的抑制而发挥促进血管平滑肌细胞增殖功能。

关键词:脂蛋白,低密度,氧化型;肌,平滑,血管;磷酸酶,同源性,细胞骨架蛋白;动脉粥样硬化

Abstract:

Aim To investigate the cellular signal transduction pathway of vascular smooth muscle cell (VSMC) proliferation stimulated by oxidized low density lipoprotein (ox LDL). Methods Rabbit aortic VSMC was cultured in 3 groups:control, low density lipoprotein (LDL), and ox LDL. Cell proliferating ability was determined by measuring cell number and mitochondrial dehydrogenase activity (MTT assay). Western blotting was used to detect the protein expression of phosphotase and tensin homology deleted on chromosome ten (PTEN). Phosphate concentration released from PTEN specific substrat diC 16 PIP 3 was assessed using green reagent method. Results ox LDL (50 mg/L) increased cell number and MD activity to 1.78～3.21 fold, but the same concentration of LDL has no such mitogenic effect. Phosphatase activity detection shew that the dephosphate action of PTEN was inhibited by ox LDL (20～50 mg/L) in a concentration dependent manner,and this inhibiting effect was at its maximum within 10 min,lasting at least for 24 h (p<0.01, vs. control group). Conclusion Ox LDL improved VSMC proliferation probably by inhibiting the lipid phosphatase activity of PTEN.

Key words:Lipoprotein, LDL, Oxidized;Muscle, Smooth, Vascular;Phosphotase, Homologous, Cytoskeleton Protein;Atherosclerosis