【摘要】：目的：探討尿毒癥毒素甲狀旁腺激素(Parathyroid hormone，PTH)是否誘導(dǎo)血管內(nèi)皮細(xì)胞是否發(fā)生內(nèi)皮細(xì)胞-間質(zhì)轉(zhuǎn)化(Endothelial-mesenchymal transition，，EndMT)及其中的分子機(jī)制。 方法：采用人主動(dòng)脈血管內(nèi)皮細(xì)胞（HAEC）培養(yǎng)至對(duì)數(shù)生長(zhǎng)期，分別加入不同劑量的全長(zhǎng)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(gè)時(shí)間點(diǎn)，對(duì)HAEC細(xì)胞的形態(tài)變化進(jìn)行觀察，并采用熒光實(shí)時(shí)定量PCR和Western blot對(duì)HAEC細(xì)胞中內(nèi)皮細(xì)胞標(biāo)志物VE-Cadherin和CD31、間充質(zhì)細(xì)胞標(biāo)志物α-SMA的mRNA轉(zhuǎn)錄水平和蛋白表達(dá)水平進(jìn)行檢測(cè)。相關(guān)分子機(jī)制的探討主要采用Western blot對(duì)HAEC細(xì)胞中TGF-β1和ILK的表達(dá)水平進(jìn)行檢測(cè)，并利用TGF-β1信號(hào)通路抑制劑SB431542、Pirfenidone和ILK抑制劑Cpd22干預(yù)PTH誘導(dǎo)細(xì)胞，對(duì)HAEC細(xì)胞的形態(tài)變化進(jìn)行觀察，以及HAEC細(xì)胞發(fā)生EndMT的相關(guān)標(biāo)志物進(jìn)行實(shí)時(shí)定量PCR和Western blot檢測(cè)。 結(jié)果：細(xì)胞形態(tài)觀察結(jié)果提示PTH可以誘導(dǎo)血管內(nèi)皮細(xì)胞HAEC細(xì)胞在形態(tài)上發(fā)生纖維化轉(zhuǎn)變。實(shí)時(shí)定量PCR和Western blot結(jié)果都表明在PTH作用下，血管內(nèi)皮細(xì)胞標(biāo)志分子VE-Cadherin和CD31表達(dá)降低；纖維細(xì)胞標(biāo)志分子α-SMA的表達(dá)則顯著升高，且呈時(shí)間和劑量依賴性。Western blot分析進(jìn)一步發(fā)現(xiàn)HAEC細(xì)胞中TGF-β1和ILK的表達(dá)水平在PTH誘導(dǎo)下同時(shí)呈時(shí)間和劑量依賴性的升高。使用信號(hào)通路抑制劑抑制TGF-β1和ILK可以部分逆轉(zhuǎn)PTH對(duì)HAEC細(xì)胞纖維化的誘導(dǎo)增加作用，包括逆轉(zhuǎn)PTH降低內(nèi)皮細(xì)胞標(biāo)志物和增加纖維化細(xì)胞標(biāo)志物的作用。 結(jié)論：PTH誘導(dǎo)可以減弱HAEC細(xì)胞的內(nèi)皮細(xì)胞特征、增強(qiáng)纖維化特征，即誘導(dǎo)HAEC細(xì)胞發(fā)生EndMT轉(zhuǎn)化，TGF-β1/ILK信號(hào)通路是PTH誘導(dǎo)HAEC細(xì)胞發(fā)生EndMT轉(zhuǎn)化的可能的分子機(jī)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(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R692.5

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