【摘要】：目的：探討尿毒癥毒素甲狀旁腺激素(Parathyroid hormone，PTH)是否誘導血管內皮細胞是否發(fā)生內皮細胞-間質轉化(Endothelial-mesenchymal transition，，EndMT)及其中的分子機制。 方法：采用人主動脈血管內皮細胞（HAEC）培養(yǎng)至對數生長期，分別加入不同劑量的全長重組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，選擇12h、24h、36h和60h共4個時間點，對HAEC細胞的形態(tài)變化進行觀察，并采用熒光實時定量PCR和Western blot對HAEC細胞中內皮細胞標志物VE-Cadherin和CD31、間充質細胞標志物α-SMA的mRNA轉錄水平和蛋白表達水平進行檢測。相關分子機制的探討主要采用Western blot對HAEC細胞中TGF-β1和ILK的表達水平進行檢測，并利用TGF-β1信號通路抑制劑SB431542、Pirfenidone和ILK抑制劑Cpd22干預PTH誘導細胞，對HAEC細胞的形態(tài)變化進行觀察，以及HAEC細胞發(fā)生EndMT的相關標志物進行實時定量PCR和Western blot檢測。 結果：細胞形態(tài)觀察結果提示PTH可以誘導血管內皮細胞HAEC細胞在形態(tài)上發(fā)生纖維化轉變。實時定量PCR和Western blot結果都表明在PTH作用下，血管內皮細胞標志分子VE-Cadherin和CD31表達降低；纖維細胞標志分子α-SMA的表達則顯著升高，且呈時間和劑量依賴性。Western blot分析進一步發(fā)現HAEC細胞中TGF-β1和ILK的表達水平在PTH誘導下同時呈時間和劑量依賴性的升高。使用信號通路抑制劑抑制TGF-β1和ILK可以部分逆轉PTH對HAEC細胞纖維化的誘導增加作用，包括逆轉PTH降低內皮細胞標志物和增加纖維化細胞標志物的作用。 結論：PTH誘導可以減弱HAEC細胞的內皮細胞特征、增強纖維化特征，即誘導HAEC細胞發(fā)生EndMT轉化，TGF-β1/ILK信號通路是PTH誘導HAEC細胞發(fā)生EndMT轉化的可能的分子機制之一。
[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 偽-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 偽 -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.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R692.5

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