【摘要】：目的：探討人腎微血管內(nèi)皮細胞—間充質(zhì)細胞轉(zhuǎn)分化(Endothelial-mesenchymal transition, EndMT)在移植腎間質(zhì)纖維化形成中的意義及相關(guān)機制。方法：通過對本院25例慢性移植腎失功能(Chronic allograft dysfunction,CAD)患者和25例正常人血生化指標、以及移植腎組織標本和正常腎組織標本進行、糖原(PAS)和馬松三色(Masson)染色檢查分析,觀察兩組研究對象腎功能、腎小管萎縮和腎小球塌陷及腎間質(zhì)纖維化程度的差異。用免疫組織化學和間接免疫熒光雙重染色方法檢測兩組腎組織標本中血管內(nèi)皮細胞標志物——CD34和肌成纖維細胞標志物——αα-平滑肌細胞肌動蛋白(α-Smooth muscle actin, α-SMA)以及轉(zhuǎn)化生長因子-β1 (Transforming growth factor-β1, TGF-β1)的表達和分布特點。進一步以原代人臍靜脈內(nèi)皮細胞(Human umbilical vein endothelial cell, HUVEC)為體外研究對象,予以TGF-β1 (5ng/m 1)分別作用0-72h,采用免疫蛋白印跡方法觀察細胞中CD34和α-SMA的表達變化。結(jié)果：與正常人相比,CAD患者血清肌酐水平明顯升高,且PAS和Masson三色染色結(jié)果顯示CAD患者移植腎組織中出現(xiàn)明顯腎小管萎縮、腎小球塌陷及腎間質(zhì)纖維化改變;進一步半定量統(tǒng)計分析結(jié)果表明兩者間存在統(tǒng)計學差異(P0.01)。免疫組織化學及間接免疫熒光染色結(jié)果表明,與正常組相比,CAD組中CD34陽性表達率降低而α-SMA及TGF-β1陽性表達率則顯著升高。且間接免疫熒光雙重染色研究發(fā)現(xiàn)CAD組中部分腎小球和間質(zhì)微血管內(nèi)皮細胞呈CD34和α-SMA雙重染色陽性。體外研究顯示,TGF-β1作用于HUVEC細胞后,隨著時間延長,CD34表達逐漸降低而(α-SMA表達逐漸增多,與對照組相比,具有統(tǒng)計學差(P0.01)。結(jié)論：人腎微血管內(nèi)皮細胞可能在TGF-β1介導下通過發(fā)生EndMT現(xiàn)象進而在移植腎間質(zhì)纖維化形成中起重要作用。
[Abstract]:Aim: to investigate the significance and mechanism of human renal microvascular endothelial cell-mesenchymal cell transdifferentiation (Endothelial-mesenchymal transition, EndMT) in the formation of renal interstitial fibrosis. Methods: the blood biochemical indexes of 25 patients with chronic renal allograft dysfunction (Chronic allograft dysfunction,CAD) and 25 normal subjects were studied. The difference of renal function, renal tubular atrophy, glomerular collapse and renal interstitial fibrosis between the two groups was observed by (PAS) and Ma Song trichrome (Masson) staining. Detection of vascular endothelial cell markers-CD34 and myofibroblast markers-偽-smooth muscle actin (偽-Smooth muscle actin,) in renal tissues by immunohistochemistry and indirect immunofluorescence double staining The expression and distribution of 偽-SMA and transforming growth factor- 尾 1 (Transforming growth factor- 尾 1, TGF- 尾 1. Furthermore, the primary human umbilical vein endothelial cells (Human umbilical vein endothelial cell, HUVEC) were treated with TGF- 尾 1 (5ng/m 1) for 0-72 h in vitro. The expression of CD34 and 偽 SMA were observed by Western blot. Results: the serum creatinine level in CAD patients was significantly higher than that in normal controls. The results of PAS and Masson trichromatic staining showed that renal tubular atrophy, glomerular collapse and interstitial fibrosis were observed in CAD patients. Further semi-quantitative statistical analysis showed that there was a statistical difference between the two (P0.01). The results of immunohistochemistry and indirect immunofluorescence staining showed that the positive expression rate of CD34 in CAD group was lower than that in normal group, but the positive rate of 偽-SMA and TGF- 尾 1 was significantly increased in CAD group. Indirect immunofluorescence double staining showed that some glomerular and interstitial microvascular endothelial cells in CAD group were double stained with CD34 and 偽-SMA. In vitro studies showed that after TGF- 尾 1 was treated with HUVEC cells, the expression of CD34 decreased gradually and the expression of 偽-SMA increased with the prolongation of time. Compared with the control group, the expression of 偽-SMA was significantly lower than that of the control group (P0.01). Conclusion: human renal microvascular endothelial cells may play an important role in the formation of renal interstitial fibrosis by EndMT phenomenon mediated by TGF- 尾 1.
【學位授予單位】：南京醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R692

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