可溶性鸟苷酸环化酶激活剂cinaciguat对高糖状态下人脐静脉内皮细胞的影响

发布时间：2018-06-23 19:01

本文选题：cinaciguat + 高糖　；参考：《山东大学》2016年硕士论文

【摘要】：研究背景糖尿病血管病变因其较高的发生率、致残率以及致死率成为糖尿病最主要的并发症之一。内皮功能损伤是糖尿病血管病变的始动环节和关键。高糖可通过多种机制损伤内皮功能,如氧化应激、炎症反应等。一氧化氮(NO)作为一种信号转导分子,能够通过NO/可溶性鸟苷酸环化酶(sGC)/环磷酸鸟苷(cGMP)信号途径发挥血管舒张、抗炎、抗氧化的作用。病理情况下,NO与sGC均被氧化,使此信号通路无法正常进行。Cinaciguat(以下简称CIN)是一种新发现的sGC激活剂,其既不依赖NO又可激活被氧化的sGC,从而激活上述通路发挥抗炎、抗氧化的特性。有研究表明其对心肌梗死、心肌缺血或再灌注损伤、氧化应激引起的内皮功能紊乱、新生血管内膜形成、以及心肌肥大等均有改善作用。但CIN在糖尿病血管病变的发病过程中的作用尚不明确。因此本研究选择高糖诱导的人脐静脉内皮细胞(HUVECs)损伤模型,模拟糖尿病的体内环境,探索CIN对糖尿病血管内皮细胞的保护作用。研究目的观察CIN对高糖环境下人脐静脉内皮细胞一氧化氮(NO)、氧化应激相关指标以及炎症相关指标的影响,评价其对糖尿病血管内皮细胞的保护作用,并初步探讨其作用机制,为糖尿病血管病变的靶向治疗提供理论依据。研究方法1.细胞来源及培养：购买的人脐静脉内皮细胞株置于含10%胎牛血清的DMEM培养基中,放入37℃、5%的C02培养箱中培养。2.高糖损伤模型的建立：根据预实验结果,采用葡萄糖浓度为33.3mmol/L作用于体外培养的HUVECs细胞48h建立高糖损伤模型。3.CIN对细胞增殖活性的影响：HUVECs细胞生长至70%-80%融合时,高糖(33.3mmol/L)孵育48h诱导损伤,用CIN(0.01、0.1、1和10μmol/L)处理细胞48h,MTT法测细胞存活率。4.实验分组：HUVECs细胞生长至70%-80%融合时,将HUVECs随机分为4组：①正常组(葡萄糖浓度为5.5mmol/L):②正常组+CIN组(葡萄糖浓度为5.5mmol/L+CIN浓度为1μmol/L)；③高糖组(葡萄糖浓度33.3mmol/L)；④高糖组+CIN组(葡萄糖浓度为33.3mmol/L+CIN浓度为1μm01/L).处理48h用于后续实验。5.采用硝酸还原酶法测NO水平。6.氧化应激相关指标的检测：利用荧光探针DCFH-DA检测ROS,采用硫代巴比妥酯法、黄嘌呤氧化酶法检测MDA.SOD的含量。7.采用实时荧光定量聚合酶链反应法(RT-PCR)检测ICAM-1、VCAM-1的mRNA表达水平。8.采用免疫印迹法检测ICAM-1、VCAM-1、NF-κ Bp65的蛋白表达水平。研究结果1.细胞存活率的检测：与正常组相比,高糖组细胞存活率降为(40.7±10.1)%,与正常组相比下降了59.3%(P0.01)。与高糖组相比,高糖+CIN(0.01、0.1、1、10κmol/L)各组的细胞活性增加为(60.0±13.6)%,(79.1±11.6)%,(93.1±9.1)%、(93.4±9.3)%,与正常组相比增加了40%、20.9%、6.9%、6.6%(均P0.05)。但CIN1μmol/L与CIN10μmol/L相比细胞活性无明显差别(P0.05)。2.NO的生成量：与正常组相比,高糖组NO生成量明显减少(18.26±2.03μmol/L vs 12.78±0.92μm01/L,P=0.000).与高糖组相比,高糖组+CIN组NO生成量明显增加(12.78±0.92μmol/L vs 17.24±1.69μmol/L,P=0.001)。与正常组相比,正常组+CIN组NO生成量无显著差异(P0.05)。3.氧化应激：与正常组相比,高糖组ROS生成量显著增加(1.00-0.00 vs 2.57±0.44,P=0.002).MDA含量也显著增加(1.32±0.37nmol/ml vs 9.32±2.21nmol/ml,P=0.001),SOD活性显著减低(22.82±4.17 U/ml vs 10.79±2.66 U/ml,P=0.000).与高糖组相比,高糖组+CIN组ROS生成量显著降低(2.57±0.44 vs 1.61±0.28,P=0.01),MDA的生成量也显著降低(9.32±2.21nmol/m1 vs 4.22±1.55 nmol/ml,P=0.007),SOD的活性显著升高(10.79±2.66 U/ml vs 18.09±3.36 U/ml,P=0.001).与正常组相比,正常组+CN组ROS、MDA生成量以及SOD活性无显著性差异(P0.05)。4.炎症相关指标：与正常组相比,高糖组ICAM-1、VCAM-1 mRNA以及蛋白表达均显著增高(P0.05), NF-KBp65蛋白表达也显著增高(P0.05)；与高糖组相比,高糖组+CIN组ICAM-1、 VCAM-1 mRNA以及蛋白表达均显著降低(P0.05)、NF-KBp65蛋白表达也显著降低(P0.05)。与正常组相比,正常组+CIN组ICAM-1, VCAM-1以及NF-KBp65表达无显著性差异(P0.05)。研究结论1.高糖可使体外培养的人脐静脉内皮细胞存活减少,而cinaciguat可逆转被高糖抑制的内皮细胞的活性,保护血管内皮细胞。2.高糖使内皮细胞存活减少的机制包括NO的生成减少、氧化应激反应增强以及炎症因子增多。3. cinaciguat可通过提高NO含量,减少氧化应激相关产物ROS和MDA的生成,提高抗氧化酶SOD的活性以及抑制炎症相关指标ICAM-1、VCAM-1、 NF-κBp65的表达发挥对内皮细胞的保护作用。
[Abstract]:The effect of CIN on vascular endothelial cells in diabetic vascular endothelial cells was studied . The effects of NO and sGC on the endothelial function , the formation of new angiogenesis and the protective effect of CIN on diabetic vascular endothelial cells were studied .
( 3 ) high glucose group ( glucose concentration : 33.3 mmol / L ) ;
The expression level of ICAM - 1 , VCAM - 1 and NF - 魏B were measured by means of RT - PCR . Results : Compared with the normal group , the content of ICAM - 1 , VCAM - 1 mRNA was decreased by 59.3 % ( P0.01 ) . Compared with high - sugar group , NO production in high - sugar group was significantly increased ( 12.78 卤 0.92 渭mol / L vs 17.24 卤 1.69 渭mol / L , P = 0.001 ) . Compared with the normal group , there was no significant difference in NO production in the normal group and the normal group ( P0.05 ) . The ROS production in the high sugar group increased significantly ( 1.00 - 0.00 vs 2.57 卤 0.44 , P = 0.002 ) compared with the normal group . The content of MDA also increased significantly ( 1.32 卤 0.37 nmol / ml vs 9.32 卤 2.21nmol / ml , P = 0.001 ) , and SOD activity was significantly decreased ( 22.82 卤 4.17 U / ml vs 10.79 卤 2.66 U / ml , P = 0.000 ) . Compared with the high - sugar group , the amount of ROS in the high - sugar group was significantly decreased ( 2.57 卤 0.44 vs 1.61 卤 0.28 , P = 0.01 ) , and the amount of MDA increased significantly ( 9.32 卤 2.21nmol / ml vs 4.22 卤 1.55 nmol / ml , P = 0.007 ) . The activities of SOD increased significantly ( 10.79 卤 2.66 U / ml vs 18.09 卤 3.36 U / ml , P = 0.001 ) . Compared with the normal group , the levels of ROS , MDA and SOD in the normal group were significantly higher than those in the normal group ( P0.05 ) .
Compared with the normal group , the expression of ICAM - 1 , VCAM - 1 mRNA and protein in high sugar group and CIN group were significantly decreased ( P0.05 ) .
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R587.2

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