甲状旁腺激素在尿毒症中诱导血管内皮细胞间质转化的作用和机制研究
发布时间：2018-10-15 11:17
【摘要】：目的：探讨尿毒症毒素甲状旁腺激素(Parathyroid hormone，PTH)是否诱导血管内皮细胞是否发生内皮细胞-间质转化(Endothelial-mesenchymal transition，，EndMT)及其中的分子机制。 方法：采用人主动脉血管内皮细胞（HAEC）培养至对数生长期，分别加入不同剂量的全长重组PTH(10-12mol/L、10-11mol/L、10-10mol/L、10-9mol/L、10-8mol/L)作用48h；或在10-8mol/L PTH作用下，选择12h、24h、36h和60h共4个时间点，对HAEC细胞的形态变化进行观察，并采用荧光实时定量PCR和Western blot对HAEC细胞中内皮细胞标志物VE-Cadherin和CD31、间充质细胞标志物α-SMA的mRNA转录水平和蛋白表达水平进行检测。相关分子机制的探讨主要采用Western blot对HAEC细胞中TGF-β1和ILK的表达水平进行检测，并利用TGF-β1信号通路抑制剂SB431542、Pirfenidone和ILK抑制剂Cpd22干预PTH诱导细胞，对HAEC细胞的形态变化进行观察，以及HAEC细胞发生EndMT的相关标志物进行实时定量PCR和Western blot检测。 结果：细胞形态观察结果提示PTH可以诱导血管内皮细胞HAEC细胞在形态上发生纤维化转变。实时定量PCR和Western blot结果都表明在PTH作用下，血管内皮细胞标志分子VE-Cadherin和CD31表达降低；纤维细胞标志分子α-SMA的表达则显著升高，且呈时间和剂量依赖性。Western blot分析进一步发现HAEC细胞中TGF-β1和ILK的表达水平在PTH诱导下同时呈时间和剂量依赖性的升高。使用信号通路抑制剂抑制TGF-β1和ILK可以部分逆转PTH对HAEC细胞纤维化的诱导增加作用，包括逆转PTH降低内皮细胞标志物和增加纤维化细胞标志物的作用。 结论：PTH诱导可以减弱HAEC细胞的内皮细胞特征、增强纤维化特征，即诱导HAEC细胞发生EndMT转化，TGF-β1/ILK信号通路是PTH诱导HAEC细胞发生EndMT转化的可能的分子机制之一。
[Abstract]:Aim: to investigate whether uremic toxin parathyroid hormone (Parathyroid hormone,PTH) induces endothelial cell-interstitial transformation (Endothelial-mesenchymal transition,EndMT) in vascular endothelial cells and its molecular mechanism. Methods: human aortic vascular endothelial cells (HAEC) were cultured to logarithmic growth stage and treated with different doses of full-length recombinant PTH (10-12 mol / L 10-11 mol / L 10 -10 mol / L 10 -9 mol / L 10 -8 mol / L) for 48 h, or 12 h, 24 h, 36 h and 60 h after 10-8mol/L PTH treatment, and the morphological changes of HAEC cells were observed. The mRNA transcription and protein expression of VE-Cadherin and CD31, mesenchymal marker 伪-SMA in HAEC cells were detected by real-time quantitative PCR and Western blot. Western blot was used to detect the expression of TGF- 尾 1 and ILK in HAEC cells, and TGF- 尾 1 signaling inhibitor SB431542,Pirfenidone and ILK inhibitor Cpd22 were used to induce PTH to observe the morphological changes of HAEC cells. The relative markers of EndMT in HAEC cells were detected by real-time quantitative PCR and Western blot. Results: the morphological observation indicated that PTH could induce morphological fibrosis of vascular endothelial HAEC cells. The results of real-time quantitative PCR and Western blot showed that the expression of VE-Cadherin and CD31 in vascular endothelial cells was decreased, and the expression of 伪 -SMA in fibroblasts was significantly increased under the action of PTH. Furthermore, the expression of TGF- 尾 1 and ILK in HAEC cells was found to be increased in a time and dose dependent manner by time and dose dependent. Western blot analysis. The inhibition of TGF- 尾 1 and ILK by signal pathway inhibitors could partially reverse the increase of PTH induced fibrosis in HAEC cells, including the reduction of endothelial cell markers and the increase of fibrotic cell markers by PTH. Conclusion: PTH can attenuate the endothelial cell characteristics and enhance the fibrosis of HAEC cells, that is, to induce EndMT transformation in HAEC cells. TGF- 尾 1/ILK signaling pathway is one of the possible molecular mechanisms of PTH induced EndMT transformation in HAEC cells.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R692.5
本文编号：2272380
[Abstract]:Aim: to investigate whether uremic toxin parathyroid hormone (Parathyroid hormone,PTH) induces endothelial cell-interstitial transformation (Endothelial-mesenchymal transition,EndMT) in vascular endothelial cells and its molecular mechanism. Methods: human aortic vascular endothelial cells (HAEC) were cultured to logarithmic growth stage and treated with different doses of full-length recombinant PTH (10-12 mol / L 10-11 mol / L 10 -10 mol / L 10 -9 mol / L 10 -8 mol / L) for 48 h, or 12 h, 24 h, 36 h and 60 h after 10-8mol/L PTH treatment, and the morphological changes of HAEC cells were observed. The mRNA transcription and protein expression of VE-Cadherin and CD31, mesenchymal marker 伪-SMA in HAEC cells were detected by real-time quantitative PCR and Western blot. Western blot was used to detect the expression of TGF- 尾 1 and ILK in HAEC cells, and TGF- 尾 1 signaling inhibitor SB431542,Pirfenidone and ILK inhibitor Cpd22 were used to induce PTH to observe the morphological changes of HAEC cells. The relative markers of EndMT in HAEC cells were detected by real-time quantitative PCR and Western blot. Results: the morphological observation indicated that PTH could induce morphological fibrosis of vascular endothelial HAEC cells. The results of real-time quantitative PCR and Western blot showed that the expression of VE-Cadherin and CD31 in vascular endothelial cells was decreased, and the expression of 伪 -SMA in fibroblasts was significantly increased under the action of PTH. Furthermore, the expression of TGF- 尾 1 and ILK in HAEC cells was found to be increased in a time and dose dependent manner by time and dose dependent. Western blot analysis. The inhibition of TGF- 尾 1 and ILK by signal pathway inhibitors could partially reverse the increase of PTH induced fibrosis in HAEC cells, including the reduction of endothelial cell markers and the increase of fibrotic cell markers by PTH. Conclusion: PTH can attenuate the endothelial cell characteristics and enhance the fibrosis of HAEC cells, that is, to induce EndMT transformation in HAEC cells. TGF- 尾 1/ILK signaling pathway is one of the possible molecular mechanisms of PTH induced EndMT transformation in HAEC cells.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R692.5
【参考文献】
相关期刊论文 前4条
1 孙晨;朱东林;吴学敏;;尿毒症毒素认识的新进展[J];河北医药;2010年08期
2 叶红波;杨妙玲;;探讨血液透析对尿毒症患者血管内皮功能的影响[J];吉林医学;2012年03期
3 李晓东;李英;丁新国;;甲状旁腺素对人肾小管上皮细胞凋亡的影响[J];免疫学杂志;2009年06期
4 陈斌;李艳华;刘杨;;尿毒症毒素研究进展[J];吉林医学;2013年22期
本文编号：2272380
本文链接：https://www.wllwen.com/yixuelunwen/mjlw/2272380.html
最近更新
教材专著
