硫化氢抗血管钙化机制研究

Vascular calcification is one of the manifestations of vascular degeneration, can cause serious cardiovascular events. The study confirmed that vascular calcification is an active, adjustable, treatable and preventable. The study found to have a phenotypic changes, it is vascular smooth muscle cell transformation secretory function of osteoblast-like phenotype of vascular calcification key cell marker expression in the process of phenotypic changes will change. Our previous experimental studies found that exogenous administration of hydrogen sulfide can reduce vascular calcification, by affecting the expression of bone cell markers play a role. The present study, in vivo and in vitro the calcification experimental model, while applying advanced experimental means of hydrogen sulfide to intervene in the process of vascular calcification using RNA interference, DNA methylation, receptor fluorescence analysis of modern molecular biology techniques and confocal microscopy the impact of the observed calcification node changes its variation of cell markers cystathionine-γ-lyase gene expression changes after calcification and hydrogen sulfide intervention marker expression, prevention and treatment of vascular calcification most explicitly good point in time, the whole cell level to clarify the mechanism of hydrogen sulfide to reduce vascular calcification, aims to further explore the mechanism for the effective prevention and treatment of vascular calcification and to find new therapeutic targets.

血管钙化是血管退化的表现之一，可以引起严重的心血管事件，研究证实血管钙化是一个主动、可调节、可治疗和可预防的过程，研究发现血管平滑肌细胞由收缩型转变成具有合成和分泌功能的成骨细胞样表型是血管钙化的关键，另外，在表型改变的过程中细胞标志物的表达会发生变化，我们前期的实验研究发现外源性给予硫化氢可以减轻血管钙化，是通过影响成骨细胞标志物的表达发挥作用。本实验采用在体和离体的钙化实验模型，同时施以硫化氢干预血管钙化过程，运用RNA干扰、DNA甲基化、受体荧光分析等现代分子生物学技术和共聚焦显微镜等先进实验手段，观察钙化发生的时间节点、细胞标志物的变化情况及其变化规律、胱硫醚-γ-裂解酶基因表达变化对钙化发生和硫化氢干预之后标志物表达的影响，明确防治血管钙化的最佳时间点，从整体和细胞水平阐明硫化氢减轻血管钙化的作用机制，旨在为有效防治血管钙化进一步探寻其发生机制和寻找新的治疗靶点。

背景：血管组织钙化是动脉粥样硬化、高血压、糖尿病血管病变、血管损伤和慢性肾病等疾病患者中共同的病理表现，是导致心脑血管事件(心肌梗死，脑卒中等)的重要危险因子。.内容：在成功复制HUSMCs钙化模型基础上，给予NaHS进行培养，对细胞Von Kossa染色、显微图像分析、碱性磷酸酶(alkaline phosphatase, ALP)活性和细胞钙含量等指标进行检测，观察各组细胞钙沉积情况；酶联免疫吸附法检测细胞培养液中转化生长因子β1(transforming growth factorβ1, TGFβ1)的活性；荧光定量PCR法检测细胞中TGFβ1 mRNA及核心结合因子1（core binding factor α1，Cbfα1）mRNA的表达；Western blot 法检测 HUSMCs中骨桥蛋白（osteopontin, OPN）表达，探讨硫化氢抗血管钙化机制。.结果：H2S可明显减少HUSMCs钙结节的聚集生长，Von Kossa染色显示细胞聚集处棕褐色钙结节较钙化组明显减轻（P＜0.05）；NaHS-低、中、高剂量组钙化细胞百分比、细胞钙含量和碱性磷酸酶活性较钙化组明显降低（P＜0.05）；NaHS-低、中、高剂量组与钙化组相比，培养液中TGFβ1的活性、细胞内TGFβ1 mRNA、Cbfα1mRNA表达及OPN表达均较钙化组减少（P＜0.05)。. 该项目共发表1篇中文核心。. 意义：故本实验在β-甘油磷酸盐诱导人脐静脉平滑肌细胞钙化的基础上，观察外源性硫化氢供体硫氢化钠是否可以抑制人脐静脉平滑肌细胞钙化的发生及对TGFβ1-Smads-Cbfa1信号通路的影响，揭示硫化氢抑制人脐静脉平滑肌细胞钙化的发生的可能机制。