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蛋白激酶Cβ2在不同血糖模式致人内皮细胞损伤中的作用
【作者】 林雪波；
【导师】 周波；
【作者基本信息】 重庆医科大学 ， 内科学， 2010， 硕士
【摘要】 目的研究蛋白激酶C(PKC)β2、PPARα在高糖诱导人脐静脉内皮细胞(HUVECs)表达血管内皮生长因子(VEGF)、血管细胞粘附分子1(VCAM-1)mRNA中的作用及相互关系。方法将培养的HUVECs分为以下8组:正常糖(NG,5 mmol/L D-葡萄糖)组、高糖(HG,25 mmol/L D-葡萄糖)组、渗透压对照组(L,NG + 20 mmol/L L-葡萄糖)组、正常糖空载体转染(NN,NG + Ad5-null)组、高糖PKCβ2转染(HB,HG+Ad5-PKCβ2)组、高糖+非诺贝特组(HF,HG + 40μmol/L非诺贝特)组、高糖PKCβ2转染+非诺贝特(HBF,HB + 40μmol/L非诺贝特)组,以上各组细胞均培养6天。另高糖孵育细胞6天后再与非诺贝特共培养20min作为HF20组。以RT-PCR检测VEGF、VCAM-1 mRNA的表达水平,用Western印迹法测定PPARα蛋白表达,采用激光共聚焦检测PKCβ2蛋白的表达和转位。结果(1)HG组VEGF、VCAM-1 mRNA表达增加,分别为NG组的1.91倍和1.56倍(P值均<0.05);HG诱导PKCβ2核转位激活,定量分析示HG组胞浆/胞核荧光强度比值较NG组降低37%(P<0.05);HB组PKCβ2核转位与HG组相比更加明显,VEGF、VCAM-1 mRNA表达亦进一步增加,分别为NG组的2.59倍和2.07倍(P值均<0.05)。(2)HG组PPARα蛋白表达较NG组减少了20%(P <0.05);与HG组相比,HF组PPARα蛋白水平上调了13%,VEGF、VCAM-1 mRNA表达则明显下调,分别为HG组的68%和74%(P值均<0.05);与HG组相比,HF20组VEGF、VCAM-1 mRNA表达差异无统计学意义。(3)HB组PPARα蛋白水平进一步下降,为HG组的78%(P <0.05)。结论高糖通过诱导HUVECs PKCβ2核转位激活,进而调控PPARα表达,增加VEGF、VCAM-1 mRNA的表达。第二部分蛋白激酶Cβ2-活性氧交互环介导高糖致人内皮细胞损伤记忆效应目的以人脐静脉内皮细胞(HUVECs)作为体外模拟高血糖记忆的研究对象,观察蛋白激酶C(PKC)β2、活性氧(ROS)对其表达血管内皮生长因子(VEGF)、血管细胞粘附分子1(VCAM-1)mRNA的影响,并探讨PKCβ2、ROS在其中可能的交互作用。方法将培养的HUVECs分为以下5组:正常糖(NG,5 mmol/L D-葡萄糖×3周)组、高糖(HG,25 mmol/L D-葡萄糖×3周)组、记忆(M,25 mmol/L D-葡萄糖×2周+5 mmol/L D-葡萄糖×1周)组、记忆PKCβ2转染(MB,25 mmol/L D-葡萄糖×2周+5 mmol/L D-葡萄糖×1周+ Ad5-PKCβ2)组、记忆+α-硫辛酸(MA,25 mmol/L D-葡萄糖×2周+5 mmol/L D-葡萄糖×1周+62.5μmol/Lα-硫辛酸)组。以RT-PCR法检测VEGF、VCAM-1 mRNA表达水平,用荧光显微镜和荧光酶标仪测定ROS水平,采用激光共聚焦检测PKCβ2蛋白的表达和转位。结果(1)HG组和M组VEGF、VCAM-1 mRNA表达增加,分别为NG组的1.76倍、1.35倍和1.69倍、1.43倍(P值均<0.05)。(2)HG组和M组ROS水平分别较NG组升高了86%、80% (P值均<0.05);MA组ROS水平较M组下降了38%,同时MA组VEGF、VCAM-1mRNA表达亦较M组显著下调,分别为M组的67%、78%(P值均<0.05)。(3)HG组和M组PKCβ2核转位激活,定量分析显示,胞浆/胞核荧光强度比值均较NG组下降,分别为NG组的70%、74%( P值均<0.05);MB组PKCβ2核转位较M组更为明显,胞浆/胞核荧光强度比值为M组的77%,同时MB组VEGF、VCAM-1 mRNA表达亦较M组进一步上调,分别为M组的1.29倍、1.18倍(P值均<0.05)。(4)MA组PKCβ2核转位较M组减少,胞浆/胞核荧光强度比值为M组的1.18倍;而MB组ROS水平较M组上调了25%(P值均<0.05)。结论HUVECs存在高糖记忆效应,PKCβ2-ROS交互环可能介导了这个效应的发生。第三部分高糖波动激活蛋白激酶Cβ2损伤人内皮细胞及二甲双胍的干预作用目的观察波动性高糖与持续性高糖对人脐静脉内皮细胞(HUVECs)血管内皮生长因子(VEGF)、血管细胞粘附分子1(VCAM-1)mRNA表达及活性氧(ROS)水平的不同影响,探讨PKCβ2在其中的作用及二甲双胍(Metformin)的干预影响。方法将培养的HUVECs分为以下5组:正常糖(NG,5 mmol/L D-葡萄糖)组、持续性高糖(SHG,15 mmol/L D-葡萄糖)组、波动性高糖(IHG,5 mmol/L D-葡萄糖与25mmol/L D-葡萄糖每24h更换一次)组、波动性高糖PKCβ2转染(IHGB,IHG + Ad5-PKCβ2)组、波动性高糖+二甲双胍(IHGM,IHG+20μmol/L二甲双胍)组。以上各组细胞均培养14天。以RT-PCR法检测细胞VEGF、VCAM-1 mRNA表达水平,用荧光酶标仪测定细胞ROS水平,采用激光共聚焦检测PKCβ2蛋白表达和转位。结果(1)IHG组和SHG组VEGF、VCAM-1 mRNA表达及ROS水平增加,分别为NG组的1.29倍、2.32倍、3.21倍和1.17倍、1.63倍、1.75倍,且IHG组增加更明显(P值均<0.05)。(2)IHG组和SHG组PKCβ2核转位激活,定量分析显示胞浆/胞核荧光强度比值较NG组分别下降了21%和26%,且IHG组下降更加明显(P值均<0.05);IHGB组PKCβ2核转位较IHG组更显著,胞浆/胞核荧光强度比值为IHG组的75%,同时IHGB组VEGF、VCAM-1mRNA表达以及ROS水平亦显著高,分别为IHG组的1.16倍、1.26倍和1.25倍(P值均<0.05)。(3)IHGM组VEGF、VCAM-1mRNA表达及ROS水平均较IHG组明显低,为IHG组的87%、76%和39%;IHGM组PKCβ2核转位亦较IHG组减少,胞浆/胞核荧光强度比值为IHG组的1.09倍(P值均<0.05)。结论波动性高糖较持续性高糖更易损伤HUVECs,这与PKCβ2活化密切相关;二甲双胍可能通过抑制PKCβ2激活,减轻波动性高糖诱导的HUVECs损伤。
【Abstract】 Objective To investigate the roles of protein kinase C (PKC)β2 and PPARαin the mRNA expression of vascular endothelial growth factor (VEGF) and vascular cell adhesion molecule-1 (VCAM-1) played in human umbilical vein endothelial cells (HUVECs) exposed to high glucose, and to explore their relationship.Methods The HUVECs were divided into eight groups: normal glucose (NG, 5 mmol/L D-glucose) group, high glucose (HG, 25 mmol/L D-glucose) group, osmotic control (L, NG + 20 mmol/L L-Glucose) group, normal glucose transfected with empty vector (NN, NG + Ad5-null) group, high glucose transfected with PKCβ2 (HB, HG + Ad5-PKCβ2) group, high glucose plus fenofibrate (HF, HG + 40μmol/L fenofibrate) group, high glucose transfected with PKCβ2 plus fenofibrate (HBF, HB + 40μmol/L fenofibrate) group. HUVECs were incubated with fenofibrate for 20 minutes as HF20 group. All cells in various groups were cultured for 6 days. The expressions of VEGF and VCAM-1 mRNA were detected by RT-PCR. PPARαprotein expression was tested by Western blot. The expression and translocation of PKCβ2 protein was observed by confocal laser scanning microscopy.Results (1) HG increased VEGF and VCAM-1 mRNA expressions, with 1.91 and 1.56 folds of NG group (both P <0.05). Meanwhile, HG induced PKCβ2 translocation from cytosol to nucleus and quantitative analysis showed the ratio of cytosol/nucleus fluorescence intensity in HG group decreased by 37% compared with NG group (P <0.05); Compared with HG group, PKCβ2 in HB group translocated more obviously from cytosol to nucleus and the mRNA expressions of VEGF and VCAM-1 in HB group further increased, with 2.59 and 2.07 folds of NG group (all P<0.05). (2)The protein expression of PPARαdecreased by 20% in HG group compared with NG group (P <0.05); Compared with HG group, PPARαexpression in HF group was 13% increased and VEGF and VCAM-1 mRNA expression in HF group decreased, with 68% and 74% of HG group (all P <0.05); There was no significant differences in the expressions of VEGF and VCAM-1 mRNA between HF20 and HG groups. (3) PPARαexpression further decreased in HB group, being 78% of HG group (P <0.05).Conclusion High glucose stimulates VEGF and VCAM-1 mRNA expressions in HUVECs via PKCβ2 activation-dependent PPARαpathway. PART 2‘HYPERGLYCEMIC MEMORY’IN HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS MEDIATED BY INTERACTING LOOP OF PROTEIN KINASE CΒ2- REACTIVE OXYGEN SPECIESObjective To investigate the roles of protein kinase C (PKC)β2 and reactive oxygen species (ROS) in the mRNA expression of vascular endothelial growth factor (VEGF) and vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs), imitating‘hyperglycemic memory’in vitro and the relationship between them.Methods The HUVECs were divided into 5 groups: normal glucose (NG, 5 mmol/L D-glucose×3 weeks) group, high glucose (HG, 25 mmol/L D-glucose×3 weeks) group, memory (M, 25 mmol/L D-glucose×2 weeks + 5 mmol/L D-glucose×1 week) group, memory transfected with PKCβ2 (MB, M + Ad5-PKCβ2) group, memory plusα-lipoic acid (MA, M + 62.5μmol/L ALA) group. The mRNA expression of VEGF and VCAM-1 was detected by RT-PCR. ROS levels was tested with fluorescence microscopy and fluorescence microplate reader. The expression and translocation of PKCβ2 protein was observed by confocal laser scanning microscopy.Results (1) VEGF and VCAM-1 mRNA expression in group HG and M increased, being 1.76, 1.35 and 1.69, 1.43 folds of those in group NG (all P <0.05). (2) ROS levels in group HG and M increased 86% and 80% as compared with those in group NG (both P <0.05); As compared with group M, ROS levels in group MA decreased 38%, and VEGF、VCAM-1 mRNA expression in group MA decreased significantly to 67% and 78% (all P <0.05). (3) PKCβ2 was activated and translocated from cytosol to nucleus in group HG and group M. Quantitative analysis showed the ratio of cytosol/nuclear fluorescence intensity in group HG and group M decreased to 70% and 74% of those in group NG (both P <0.05); As compared with group M, PKCβ2 in group MB translocated more obviously and the mRNA expression of VEGF and VCAM-1 in group MB significantly increased 1.29 and 1.18 folds of those in group M (both P<0.05). (4) As compared with group M, the activation of PKCβ2 in group MA was inhibited and the ratio of cytosol/nuclear fluorescence intensity increased 18%. Besides, ROS levels in group MB was 25% up-regulated as compared with those in group M (both P <0.05).Conclusion Persistence of‘hyperglycemic memory’exists after glucose levels are normalized in HUVECs. This may be related with the interacting loop of PKCβ2–ROS.PART 3 INTERMITTENT HIGH GLUCOSE ENHANCES HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS INJURY THROUGH PKCΒ2 ACTIVATION AND THE EFFECT OF METFORMINObjective To investigate the role of protein kinase C (PKC)β2 in vascular endothelial growth factor (VEGF), vascular cell adhesion molecule-1(VCAM-1) mRNA expression and reactive oxygen species (ROS) leves in human umbilical vein endothelial cells (HUVECs) exposed to intermittent high glucose and the effect of Metformin. Methods The HUVECs were divided into five groups: normal glucose (NG, 5 mmol/L D-glucose) group, stable high glucose (SHG, 15 mmol/L D-glucose) group, Intermittent high glucose (IHG, alternating 5 mmol/L D-glucose and 25 mmol/L D-glucose every 24h) group, Intermittent high glucose transfected with PKCβ2 (IHGB, IHG+Ad5-PKCβ2) group, Intermittent high glucose plus metformin (IHGM, IHG+20μmol/L metformin) group. After 14 days, the mRNA expression of VEGF and VCAM-1 were detected with RT-PCR. ROS levels was tested with fluorescence microplate reader. The expression and translocation of PKCβ2 protein was observed by confocal laser scanning microscopy.Results (1) VEGF, VCAM-1 mRNA expression and ROS levels in group IHG and SHG increased, being 1.29, 2.32, 3.21 and 1.17, 1.63, 1.75 folds of those in group NG (all P <0.05) and this increase was more marked in group IHG (P<0.05). (2) PKCβ2 was activated and translocated from cytosol to nucleus in group IHG and group SHG. Quantitative analysis showed the ratio of cytosol/nucleus fluorescence intensity in group IHG and SHG was 21% and 26% decreased as compared with those in group NG, but in group IHG the decrease was more marked (all P<0.05). As compared with group IHG, PKCβ2 in group IHGB translocated more obviously and VEGF, VCAM-1 mRNA expression and ROS levels in group IHGB increased 1.16, 1.26 and 1.25 folds of those in group IHG (all P<0.05). (3) As compared with group IHG, VEGF, VCAM-1 mRNA expression and ROS levels in group IHGM decreased significantly to 87%, 76% and 39%, and the activation of PKCβ2 in group IHGM was inhibited with the ratio of cytosol/nuclear fluorescence intensity increase 1.09 fold as compared with that in group IHG (all P<0.05).Conclusion Intermittent high glucose and stable high glucose induce severe injury of HUVECs, especially IHG, which is closely associated with PKCβ2 activation. Metformin could alleviate HUVECs injury induced by intermittent high glucose by blocking PKCβ2 activation.