縫隙連接對華法令誘導的大鼠血管鈣化的作用及機制研究
發(fā)布時間：2019-03-27 17:26
【摘要】：背景:血管鈣化(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個重要的角色,目前大家比較認同的VC危險因素有:高血壓,糖尿病,年齡,慢性腎臟疾病,吸煙和炎癥。華法令是目前最常用的口服抗凝劑之一,屬于維生素K拮抗劑,目前我們已經(jīng)知道華法令的使用會增加機體VC的可能性,其原理是因為華法令抑制了體內(nèi)基質Gla蛋白(Matrix Gla protein,MGP)的形成而導致的,而MGP是體內(nèi)最強的VC抑制劑之一。迄今為止,有關于VC的發(fā)生機制依然未研究透徹,因此對其治療依然無十分有效的藥物,目前更多研究學者偏向于認為VC是由于血管平滑肌細胞(Vascular Smooth Muscle Cell,VSMC)向成骨樣細胞表型轉化而導致的�？p隙連接(Gap Junction,GJ)是除血細胞和骨骼肌細胞間外廣泛存在于其他組織細胞間的一種細胞連接形式,它在傳遞細胞間化學信息,協(xié)調(diào)細胞的代謝活動,調(diào)控細胞的生長和分化中發(fā)揮重要作用。并且GJ介導的細胞間通訊直接影響VSMCs的表型轉化。因此在本試驗中我們研究GJ在VC發(fā)生中的作用。方法:把大鼠隨機分為三組,分別為:對照組,鈣化組,甘珀酸(Carbenoxolone,CBX)干預組,其中鈣化模型組的大鼠給予注射華法令和維生素K1,CBX干預組是在鈣化模型組的基礎上加入CBX,實驗結束后,用Von Kossa染色鏡下觀察各組大鼠動脈鈣化的情況,用鈣含量試劑盒測定各組大鼠動脈的鈣沉積含量,用堿性磷酸酶(Alkaline Phosphatase,ALP)試劑盒測定大鼠血清中ALP活性,采用熒光定量PCR法和Western Blot法檢測連接蛋白43(Connexin,Cx43),Runx2,α-SMactin的mRNA和蛋白表達水平變化。結果:1、對照組的大鼠胸主動脈中未見黑色顆粒,鈣化模型組則可見胸主動脈的中膜有大量黑色顆粒,CBX干預組的鏡下所見則處于前兩者之間,大鼠胸主動脈組織的中膜依然可見散在的黑色顆粒,但相對鈣化模型組已有明顯好轉。2、對比實驗對照組,華法令可以顯著提升大鼠動脈鈣沉積含量(P0.05),而與鈣化模型組相比較,CBX干預組的鈣沉積含則量明顯下降(P0.05)3、鈣化組內(nèi)ALP的活性明顯高于對照組(P0.05),加入CBX后,ALP的活性明顯下降(P0.05)。4、對比空白對照組華法令可以顯著提升大鼠主動脈內(nèi)Cx43、Runx2 mRNA和蛋白的表達(P0.05),加入CBX后與鈣化模型組相比大鼠主動脈內(nèi)Cx43、Runx2mRNA和蛋白的表達明顯下降(P0.05),而α-SMactin的情況恰好相反。結論:本研究提示,CBX可能通過抑制Cx43的表達來抑制VSMC向成骨細胞的轉化從而抑制動脈鈣化的發(fā)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, 偽-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 偽-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.
【學位授予單位】：南昌大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R54
本文編號：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, 偽-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 偽-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.
【學位授予單位】：南昌大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R54
【參考文獻】
相關期刊論文 前1條
1 范書英;柯元南;曾玉杰;蘇德淳;王勇;張培;;不同β受體阻滯劑對大鼠心肌間隙連接結構作用的對比研究[J];中華心血管病雜志;2007年02期
，本文編號：2448389
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