缝隙连接对华法令诱导的大鼠血管钙化的作用及机制研究
发布时间：2019-03-27 17:26
【摘要】：背景:血管钙化(Vascular Calcification,VC)在冠心病,缺血卒中,外周血管疾病的病理生理中扮演一个重要的角色,目前大家比较认同的VC危险因素有:高血压,糖尿病,年龄,慢性肾脏疾病,吸烟和炎症。华法令是目前最常用的口服抗凝剂之一,属于维生素K拮抗剂,目前我们已经知道华法令的使用会增加机体VC的可能性,其原理是因为华法令抑制了体内基质Gla蛋白(Matrix Gla protein,MGP)的形成而导致的,而MGP是体内最强的VC抑制剂之一。迄今为止,有关于VC的发生机制依然未研究透彻,因此对其治疗依然无十分有效的药物,目前更多研究学者偏向于认为VC是由于血管平滑肌细胞(Vascular Smooth Muscle Cell,VSMC)向成骨样细胞表型转化而导致的。缝隙连接(Gap Junction,GJ)是除血细胞和骨骼肌细胞间外广泛存在于其他组织细胞间的一种细胞连接形式,它在传递细胞间化学信息,协调细胞的代谢活动,调控细胞的生长和分化中发挥重要作用。并且GJ介导的细胞间通讯直接影响VSMCs的表型转化。因此在本试验中我们研究GJ在VC发生中的作用。方法:把大鼠随机分为三组,分别为:对照组,钙化组,甘珀酸(Carbenoxolone,CBX)干预组,其中钙化模型组的大鼠给予注射华法令和维生素K1,CBX干预组是在钙化模型组的基础上加入CBX,实验结束后,用Von Kossa染色镜下观察各组大鼠动脉钙化的情况,用钙含量试剂盒测定各组大鼠动脉的钙沉积含量,用碱性磷酸酶(Alkaline Phosphatase,ALP)试剂盒测定大鼠血清中ALP活性,采用荧光定量PCR法和Western Blot法检测连接蛋白43(Connexin,Cx43),Runx2,α-SMactin的mRNA和蛋白表达水平变化。结果:1、对照组的大鼠胸主动脉中未见黑色颗粒,钙化模型组则可见胸主动脉的中膜有大量黑色颗粒,CBX干预组的镜下所见则处于前两者之间,大鼠胸主动脉组织的中膜依然可见散在的黑色颗粒,但相对钙化模型组已有明显好转。2、对比实验对照组,华法令可以显著提升大鼠动脉钙沉积含量(P0.05),而与钙化模型组相比较,CBX干预组的钙沉积含则量明显下降(P0.05)3、钙化组内ALP的活性明显高于对照组(P0.05),加入CBX后,ALP的活性明显下降(P0.05)。4、对比空白对照组华法令可以显著提升大鼠主动脉内Cx43、Runx2 mRNA和蛋白的表达(P0.05),加入CBX后与钙化模型组相比大鼠主动脉内Cx43、Runx2mRNA和蛋白的表达明显下降(P0.05),而α-SMactin的情况恰好相反。结论:本研究提示,CBX可能通过抑制Cx43的表达来抑制VSMC向成骨细胞的转化从而抑制动脉钙化的发生。
[Abstract]:Background: vascular calcification (Vascular Calcification,VC) plays an important role in pathophysiology of coronary heart disease, ischemic stroke and peripheral vascular disease. Chronic kidney disease, smoking and inflammation. Warfarin is one of the most commonly used oral anticoagulants, belonging to vitamin K antagonists. At present, we already know that warfarin will increase the possibility of VC in the body. The principle of warfarin is that warfarin inhibits the matrix Gla protein (Matrix Gla protein, in the body. MGP), and MGP is one of the strongest inhibitors of VC in vivo. So far, the mechanism of VC has not been thoroughly studied, so there are still no effective drugs for the treatment of VC. At present, more researchers tend to think that VC is due to vascular smooth muscle cell (VSMC) (Vascular Smooth Muscle Cell,. The transformation of VSMC to osteoblast-like cell phenotype results in the transformation of osteoblast-like cells into osteoblasts. Gap junction (Gap Junction,GJ) is a kind of cell junctions that exist widely in other tissues except blood cells and skeletal muscle cells. It transmits chemical information between cells and coordinates the metabolic activities of cells. It plays an important role in regulating the growth and differentiation of cells. Moreover, GJ-mediated intercellular communication directly affects the phenotypic transformation of VSMCs. Therefore, in this experiment, we study the role of GJ in the occurrence of VC. Methods: rats were randomly divided into three groups: control group, calcification group and Carbenoxolone,CBX intervention group, in which rats in calcification model group were injected with warfarin and vitamin K1, After the calcification model group was added to the CBX intervention group, the calcification of the arteries of each group was observed by Von Kossa staining microscope, and the calcium deposition content of the arteries of each group was measured by calcium content kit, and the calcium deposition of the arteries of the rats in each group was measured by using the Von Kossa staining microscope. Serum ALP activity was measured by alkaline phosphatase (Alkaline Phosphatase,ALP) kit, mRNA and protein expression levels of Connexin,Cx43, Runx2, 伪-SMactin were measured by fluorescence quantitative PCR and Western Blot. Results: 1. No black particles were found in the thoracic aorta of the control group, a large number of black particles were found in the media of the thoracic aorta in the calcification model group, and the first two were seen under the microscope in the CBX intervention group. In contrast to the experimental control group, warfarin could significantly increase the content of calcium deposition in the arteries of rats (P0.05), but the scattered black particles could still be seen in the media of the thoracic aorta of the rats, but the calcification model group was obviously better than the control group (P0.05). Compared with the calcification model group, the calcium deposition content in the CBX intervention group decreased significantly (P0.05), and the activity of ALP in the calcified group was significantly higher than that in the control group (P0.05). After adding CBX, the activity of ALP decreased significantly (P0.05). Compared with the blank control group, Warfarin significantly increased the expression of Cx43,Runx2mRNA and protein in rat aorta (P0.05). Compared with the calcified model group, the expression of Cx43,Runx2mRNA and protein in aorta decreased significantly after adding CBX (P0.05). The case for 伪-SMactin is the opposite. Conclusion: this study suggests that CBX may inhibit the transformation of VSMC into osteoblasts by inhibiting the expression of Cx43 and thus inhibit the occurrence of arterial calcification.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R54
本文编号：2448389
[Abstract]:Background: vascular calcification (Vascular Calcification,VC) plays an important role in pathophysiology of coronary heart disease, ischemic stroke and peripheral vascular disease. Chronic kidney disease, smoking and inflammation. Warfarin is one of the most commonly used oral anticoagulants, belonging to vitamin K antagonists. At present, we already know that warfarin will increase the possibility of VC in the body. The principle of warfarin is that warfarin inhibits the matrix Gla protein (Matrix Gla protein, in the body. MGP), and MGP is one of the strongest inhibitors of VC in vivo. So far, the mechanism of VC has not been thoroughly studied, so there are still no effective drugs for the treatment of VC. At present, more researchers tend to think that VC is due to vascular smooth muscle cell (VSMC) (Vascular Smooth Muscle Cell,. The transformation of VSMC to osteoblast-like cell phenotype results in the transformation of osteoblast-like cells into osteoblasts. Gap junction (Gap Junction,GJ) is a kind of cell junctions that exist widely in other tissues except blood cells and skeletal muscle cells. It transmits chemical information between cells and coordinates the metabolic activities of cells. It plays an important role in regulating the growth and differentiation of cells. Moreover, GJ-mediated intercellular communication directly affects the phenotypic transformation of VSMCs. Therefore, in this experiment, we study the role of GJ in the occurrence of VC. Methods: rats were randomly divided into three groups: control group, calcification group and Carbenoxolone,CBX intervention group, in which rats in calcification model group were injected with warfarin and vitamin K1, After the calcification model group was added to the CBX intervention group, the calcification of the arteries of each group was observed by Von Kossa staining microscope, and the calcium deposition content of the arteries of each group was measured by calcium content kit, and the calcium deposition of the arteries of the rats in each group was measured by using the Von Kossa staining microscope. Serum ALP activity was measured by alkaline phosphatase (Alkaline Phosphatase,ALP) kit, mRNA and protein expression levels of Connexin,Cx43, Runx2, 伪-SMactin were measured by fluorescence quantitative PCR and Western Blot. Results: 1. No black particles were found in the thoracic aorta of the control group, a large number of black particles were found in the media of the thoracic aorta in the calcification model group, and the first two were seen under the microscope in the CBX intervention group. In contrast to the experimental control group, warfarin could significantly increase the content of calcium deposition in the arteries of rats (P0.05), but the scattered black particles could still be seen in the media of the thoracic aorta of the rats, but the calcification model group was obviously better than the control group (P0.05). Compared with the calcification model group, the calcium deposition content in the CBX intervention group decreased significantly (P0.05), and the activity of ALP in the calcified group was significantly higher than that in the control group (P0.05). After adding CBX, the activity of ALP decreased significantly (P0.05). Compared with the blank control group, Warfarin significantly increased the expression of Cx43,Runx2mRNA and protein in rat aorta (P0.05). Compared with the calcified model group, the expression of Cx43,Runx2mRNA and protein in aorta decreased significantly after adding CBX (P0.05). The case for 伪-SMactin is the opposite. Conclusion: this study suggests that CBX may inhibit the transformation of VSMC into osteoblasts by inhibiting the expression of Cx43 and thus inhibit the occurrence of arterial calcification.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R54
【参考文献】
相关期刊论文 前1条
1 范书英;柯元南;曾玉杰;苏德淳;王勇;张培;;不同β受体阻滞剂对大鼠心肌间隙连接结构作用的对比研究[J];中华心血管病杂志;2007年02期
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