二叶式主动脉瓣中miRNA-195调控瓣膜钙化的机制研究

发布时间：2018-07-09 09:35

本文选题：二叶式主动脉瓣 + 瓣膜钙化　；参考：《南京医科大学学报(自然科学版)》2017年09期

【摘要】：目的 :探究二叶式主动脉瓣中miRNA-195调控瓣膜钙化的可能机制。方法 :收集35例接受主动脉瓣置换术患者狭窄的二叶式或三叶式主动脉瓣,通过PCR及Western blot分别检测miRNA-195的表达量、果蝇母源抗皮肤生长因子(drosophila mothers against decapentaplegic 7,SMAD7)的mRNA及蛋白表达量。双荧光素酶法预测miRNA-195的靶基因。在猪瓣膜间质细胞中分别沉默和过表达miRNA-195,检测SMAD7的mRNA表达水平并探究两者的相互关系。在人类瓣膜组织上通过免疫组化染色检测基质金属蛋白酶(matrix metalloproteinase,MMP)-2、MMP-9蛋白水平,通过天狼星红染色检测胶原蛋白水平,通过Von Kossa染色比较钙化程度差异,进而研究钙化相关的功能改变。结果 :与狭窄的三叶式主动脉瓣相比,狭窄的二叶式主动脉瓣中miRNA-195表达降低,SMAD7的mRNA和蛋白水平升高。双荧光素酶实验提示SMAD7是miRNA-195的直接靶标。在猪瓣膜间质细胞中沉默miRNA-195引起SMAD7 mRNA水平升高,过表达miRNA-195引起SMAD7 mRNA水平降低。狭窄钙化的二叶式主动脉瓣组织中MMP-2、MMP-9及胶原的表达较狭窄钙化的三叶式主动脉瓣升高。结论:在二叶式主动脉瓣中,下调的miRNA-195促进了SMAD7表达,进而促进细胞外基质的纤维化并最终促进其钙化。
[Abstract]:Aim: to explore the possible mechanism of miRNA-195 regulating valve calcification in two-leaf aortic valve. Methods: the expression of miRNA-195 and the expression of (drosophila mothers against decapentaplegic 7 SMAD7 were detected by PCR and Western blot in 35 patients with stenosis of aortic valve. The target gene of miRNA-195 was predicted by double luciferase method. MiRNA-195 was silenced and overexpressed in porcine valvular interstitial cells to detect the mRNA expression of SMAD7 and to explore the relationship between them. The levels of matrix metalloproteinase (MMP) -2MMP-9, collagen and calcification were detected by immunohistochemical staining, Sirius red staining and Von Kossa staining respectively. Furthermore, the functional changes associated with calcification were studied. Results: the expression of miRNA-195 in the stenosis two-leaf aortic valve decreased the mRNA and protein levels of SMAD7 compared with the stenosis trefoil aortic valve. Double luciferase assay suggested that SMAD 7 was a direct target for miRNA-195. Silencing miRNA-195 increased the level of SMAD7 mRNA in porcine valve interstitial cells and decreased the level of SMAD7 mRNA induced by overexpression of miRNA-195. The expression of MMP-2, MMP-9 and collagen in two-lobe aortic valve with stenosis calcification was higher than that in trefoil aortic valve with narrow calcification. Conclusion: in two-leaf aortic valve, down-regulated miRNA-195 promotes the expression of SMAD7, thus promotes the fibrosis of extracellular matrix and ultimately promotes its calcification.
【作者单位】：南京医科大学第一附属医院心脏大血管外科;
【基金】：江苏省自然科学基金(BK20151590)
【分类号】：R654.2

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