姜黄素抑制大鼠血管平滑肌细胞钙化的凋亡信号机制研究

发布时间：2018-04-15 08:31

  本文选题：血管钙化 + 姜黄素　； 参考：《中山大学》2016年硕士论文

【摘要】：研究背景:血管钙化(vascular calcification)指的是钙盐沉积于动脉管壁的过程,它可以导致动脉管壁变硬,顺应性降低,在临床上常见于动脉粥样硬化,慢性肾病和糖尿病患者,严重危害人类的健康。然而,由于对血管钙化的发生和发展机制缺乏深入的认识,目前临床上缺乏治疗血管钙化的有效方法。因此,研究血管钙化的病因和发生发展的病理机制,阐明调节血管钙化的分子机制非常重要。近些年来的大量研究发现:血管钙化的过程类似于骨的生成,同样,也是一种受基因表达调控的,主动的过程。血管钙化的主要细胞来源是血管平滑肌细胞(vascular smooth muscle cells,VSMCs)。在损伤因素如钙磷代谢紊乱、氧化应激的刺激下,血管平滑肌细胞可从正常的收缩表型转换为成骨样表型并分泌大量的骨相关蛋白:碱性磷酸酶(alkaline phosphatase,ALP)、同源盒基因(Msx2)、核心结合因子α1(cbfa1/Runx2)、骨形态发生蛋白(bone morphogenetic proteins,BMPs)、成骨相关转录因子(Osterix)、骨钙素(OCN)等,从而促进细胞钙化。因此血管平滑肌细胞的成骨样分化过程与血管钙化的有着密不可分的关系。大量研究已证实了钙磷代谢紊乱是导致血管平滑肌细胞钙化和凋亡的主要因素之一。血管平滑肌细胞凋亡在血管钙化的过程中发挥了重要作用。在血管平滑肌钙化之前可检测到凋亡小体的出现,血管平滑肌细胞释放凋亡小体可启动血管钙化,细胞钙化受血管平滑肌细胞凋亡调节,通过抑制血管平滑肌细胞的凋亡可抑制其钙化的程度。我们以前的研究也证实:平滑肌细胞凋亡可调节氧化型低密度脂蛋白诱导的细胞钙化。姜黄素(Curcumin),是一种来自植物姜黄的提取物,可减轻血管病变包括血管损伤后的内膜新生和动脉粥样硬化。姜黄素对血管平滑肌细胞具有不同的药理作用:抗炎,抗氧化,抑制细胞增殖和迁移等。此外,姜黄素可抑制细胞凋亡和成骨细胞的钙化。然而,迄今为止,姜黄素对血管平滑肌细胞的成骨样分化和钙化的影响未见报道。我们推测姜黄素可能通过抑制血管平滑肌细胞凋亡来影响血管平滑肌细胞成骨样分化及钙化。本研究采用体外血管钙化模型,采用高钙高磷处理体外培养的血管平滑肌细胞,诱导细胞钙化,并用姜黄素处理细胞,观察姜黄素对血管平滑肌细胞成骨样分化和钙化的影响及其凋亡信号机制。目的:1.姜黄素是否抑制血管平滑肌细胞成骨样分化和钙化。2.姜黄素是否抑制血管平滑肌细胞凋亡。3.姜黄素抑制血管平滑肌细胞钙化的凋亡信号通路是否与JNK/Bax信号通路有关。方法:本研究采用10m M BGP和3m M氯化钙处理血管平滑肌细胞3天和10天后,用茜素红染色测细胞钙化,邻甲酚酞络合剂法测钙离子浓度,检测ALP活性。用q PCR检测血管平滑肌细胞成骨样分化的相关分子和凋亡基因的表达水平。用Western blot检测JNK/Bax的表达水平。用流式细胞技术和Caspase3活性检测的方法检测了血管平滑肌细胞凋亡。结果:姜黄素可抑制高钙高磷诱导的血管平滑肌细胞成骨样分化和钙化,同时可抑制血管平滑肌细胞凋亡。另外,姜黄素可下调p-JNK、Bax的表达水平。抑制JNK信号能明显阻断血管平滑肌细胞成骨样分化和钙化。结论:姜黄素抑制了血管平滑肌细胞成骨样分化和钙化,其机制很可能与JNK/Bax凋亡信号有关。
[Abstract]:Background: vascular calcification (vascular calcification) refers to the process of calcium deposition in the arterial wall, it can lead to arterial wall hardening, reduced compliance in clinical common in atherosclerosis, chronic kidney disease and diabetes, serious harm to human health. However, because of the occurrence and development mechanism of vascular calcification the lack of deep understanding of the current clinical lack of effective method for the treatment of vascular calcification. Therefore, the etiology of vascular calcification and the occurrence and development of pathological mechanism, to elucidate the molecular mechanisms regulating vascular calcification is very important. A large number of studies in recent years found that the generation process of vascular calcification is similar to bone of the same, is also a the regulation of gene expression, active process. The main source of cells for vascular calcification in vascular smooth muscle cells (vascular smooth muscle cells, VSMCs). The damage factors such as calcium and phosphorus generation Xie disorder, oxidative stress stimulation, vascular smooth muscle cells from normal contractile phenotype into osteoblast like phenotype and secretion of bone related protein A: alkaline phosphatase (alkaline phosphatase, ALP), homeobox gene (Msx2), core binding factor alpha 1 (cbfa1/ Runx2), bone morphogenetic protein (bone morphogenetic proteins, BMPs), bone related transcription factor (Osterix), osteocalcin (OCN), so as to promote cell calcification. Therefore bone differentiation and vascular calcification into vascular smooth muscle cells has a close relationship. A large number of studies have proved that the metabolism of calcium and phosphorus is one of the main causes of vascular calcification and apoptosis smooth muscle cells. The apoptosis of vascular smooth muscle cells play an important role in the process of vascular calcification in vascular smooth muscle. Before calcification can be detected by the appearance of apoptotic bodies, vascular smooth muscle cells to release. Dead bodies can be initiated by cell vascular calcification, calcification of vascular smooth muscle cell apoptosis regulation by inhibiting the apoptosis of vascular smooth muscle cells can inhibit the calcification. Our previous studies have confirmed that apoptosis of smooth muscle cells can regulate the oxidized low density lipoprotein induced cell calcification. Curcumin (Curcumin), is a kind of plant from turmeric the extract can reduce the vascular lesions including neointima after vascular injury and atherosclerosis. Curcumin has different pharmacological effects on vascular smooth muscle cells: anti-inflammatory, antioxidant, inhibit the proliferation and migration of cells. In addition, curcumin can inhibit the apoptosis of osteoblasts and calcification. However, so far, the effect of curcumin on vascular smooth muscle cells the differentiation and calcification of bone like effects have not been reported. We speculate that curcumin may influence by inhibiting vascular smooth muscle cell apoptosis Vascular smooth muscle cell differentiation and calcification of bone samples. This study used vascular calcification of vascular smooth muscle cells by in vitro model, high calcium and high phosphorus treatment in vitro, induce cell calcification, and cells treated with curcumin, the effects of curcumin on vascular smooth muscle cells of osteogenic differentiation and calcification effect and apoptosis signaling mechanism. Objective: 1. whether curcumin inhibits vascular smooth muscle cells is the apoptosis signaling pathway of bone like differentiation and calcification of.2. whether curcumin inhibited the apoptosis of vascular smooth muscle cells.3. curcumin inhibits calcification in vascular smooth muscle cells associated with the JNK/ Bax pathway. Methods: This study used 10m M BGP and 3M M calcium chloride treatment of vascular smooth muscle cells in 3 days and 10 days were measured calcification by alizarin red staining, measured the concentration of calcium ion o-cresolphthalein complexone method, ALP activity detection. PCR detection of vascular smooth muscle cells Q osteoblast like points The expression level of apoptosis related molecules and genes. The expression level of Western blot in the detection of JNK/Bax. The apoptosis of vascular smooth muscle cells were detected by flow cytometry and Caspase3 assay. Results: Curcumin could inhibit vascular smooth muscle cells induced by high calcium and high phosphorus into differentiation and calcification of bone, and can inhibit the apoptosis of vascular the smooth muscle cells. In addition, curcumin suppressed the p-JNK expression level of Bax. The inhibition of JNK signaling can markedly inhibit vascular smooth muscle cell differentiation and calcification of bone samples. Conclusion: Curcumin inhibits vascular smooth muscle cell differentiation and calcification of bone, its mechanism may be related to the apoptosis of JNK/Bax signal.

【学位授予单位】：中山大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R543
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本文编号：1753357