血管内皮细胞及平滑肌细胞25-羟维生素D1-a羟化酶对血管钙化的影响

The dynamic equilibrium between endothelial cell(ECs) and vascular smooth muscle cells(VSMCs）play a very important role in the formation process of vascular calcification and atherosclerosis(AS). We found that the vitamin D receptor (VDR) and ECs function abnormalities were related with the development of AS. Expression and activity of 25 - hydroxyvitamin D 1-alpha-hydroxylase (CYP27B1) in ECs and VSMCs can affect the secretion of local 1,25(OH)2D3 which it would reduce VDR activation, and promote vascular calcification. It is a very important animal model of apolipoprotein(apo) E knockout mice to study the pathogenesis of AS and vascular calcification. We isolated ECs and VSMCs in aortic from the wild type mice and apoE-/- mice respectively, then, four kinds of cells will be interacted with co-cultured. Observe the CYP27B1 expression and proliferation, apoptosis in ECs and VSMCs while they were intervened through application of parathyroid hormone(PTH) and/or 1,25(OH)2D3. Vascular inflammation and reactive excessive oxygen species(ROS) could impact on vascular calcification and the formation of AS, we detection the expression of NOX in ECs and VSMCs, observed the effect of 1,25(OH)2D3 on ROS, and it were further verificated through application NO, ROS inhibitors. Then apoE-/- mice would be injected with 1,25(OH)2D3 or PTH+1,25(OH)2D3 respectively, We would observed CYP27B1 expression and proliferation in aortic endothelial cells and VSMCs, cell and vascular calcification，in order to explore the possible mechanism which the effect of CYP27B1expression on vascular calcification in apoE-/- mice. Our aim is seeking for the hereditary basis of vascular calcification and AS, in order to find a novel pathway to prevent and treat them.

内皮细胞(ECs)平滑肌细胞(VSMCs)动态平衡参与血管钙化(VC)及动脉粥样硬化(AS)，维生素D受体(VDR)影响ECs及AS，ECs、VSMCs 25-羟维生素D1-α-羟化酶(CYP27B1)影响局部1,25(OH)2D3及VDR，促进VC。拟分离wild type鼠及AS模型-载脂蛋白(apo)E缺乏鼠主动脉ECs、VSMCs并交互共培养，甲状旁腺素(PTH)和/或1,25(OH)2D3干预，观察ECs-VSMCs相互作用及CYP27B1影响VC。活性氧(ROS)影响VC，拟查ECs、VSMCs Nox表达，观察1,25(OH)2D3影响ROS，NO、ROS酶抑制剂影响CYP27B1、细胞增生及VC，鼠体内注射1,25(OH)2D3、PTH+1,25(OH)2D3，观察ECs和VSMCs CYP27B1、细胞增生及VC。探讨ECs、VSMCs动态平衡及CYP27B1对VC影响

内皮细胞(ECs)、平滑肌细胞(VSMCs)动态平衡参与血管钙化(VC)及动脉粥样硬化(AS)，维生素D受体(VDR)影响ECs及AS，ECs、VSMCs、25-羟维生素D1α羟化酶(CYP27B1)影响局部1,25(OH)2D3及VDR，促进VC。首先我们成功分离wild type（WT）鼠及AS模型-载脂蛋白(apo)E缺乏鼠主动脉ECs、VSMCs，利用qPCR与Western-Blotting 法检测其CYP27B1基因与蛋白表达，发现WT小鼠主动脉内皮细胞CYP27B1基因表达、蛋白表达明显高于apoE-/-小鼠, 平滑肌细胞CYP27B1基因表达、蛋白表达明显降低。应用real time PCR 、WB测定测定发现WT小鼠主动脉骨桥蛋白、骨形态发生蛋白２的mRNA表达水平及蛋白表达水平均明显低于apoE-/-小鼠，而骨保护素、基质G1ａ蛋白mRNA表达水平及蛋白表达水平均明显增高。WT小鼠主动脉血管钙含量、碱性磷酸酶活性明显低于apoE-/-小鼠。将EC与VSMCs分成以下4组：apoE-/- - EC+ poE-/- －VSMCs 组（简称A组）、WT- EC+poE-/- －VSMCs 组（简称B组）、WT- EC+WT－VSMCs 组(简称C组)、apoE-/- - EC+ WT －VSMCs 组(简称D组)，置于Transwell小室中进行共培养24h、48h、72h。收集细胞进行检测。应用qPCR 与Western-Blotting 法检测各细胞内CYP27B1 基因与蛋白表达。结果发现VSMCs中apoE缺乏时，也可导致正常的EC中CYP27B1 基因表达下调，血管钙化风险增高。Western-Blotting 法检测各细胞内CYP27B1 蛋白表达，各组细胞共培养时，随着时间的延长，EC、VSMCs中CYP27B1蛋白表达与基因表达变化是一致的。利用流式细胞术检测72小时时各组细胞增生与凋亡情况，发现72小时后，apoE-/- - EC+、a poE-/- －VSMCs 组中EC、VSMCs凋亡率最高。72小时末，apoE-/-的EC与VSMCs细胞内钙含量及碱性磷酸酶活性显著增高。apoE-/-的EC与WT-VSMCs共培养时，WT-VSMCs细胞内钙含量及碱性磷酸酶活性高于WT-EC与WT-VSMCs共培养时WT-VSMCs细胞内钙含量