本文選題：苯扎氯銨 + 結膜下組織纖維化��； 參考：《廈門大學》2014年碩士論文

【摘要】：目的：苯扎氯銨(benzalkonium chloride, BAC)是滴眼液中最常用的防腐劑。大量臨床及實驗研究表明,長期使用含BAC的滴眼液會引起一系列的眼表不良反應。本研究主要探討B(tài)AC致結膜下組織纖維化的機制。 方法：SD雄性大鼠45只,隨機分成對照組、實驗組和細胞培養(yǎng)組,每組15只。對照組和實驗組分別用PBS和0.01%BAC滴左眼,每天兩次,每12小時一次。一個月后,取大鼠左眼球結膜組織提取蛋白和RNA及制備石蠟包埋切片；細胞培養(yǎng)組取大鼠左眼球結膜成纖維細胞(CFs)進行原代培養(yǎng),分別用PBS、0.00005%BAC、0.000075%BAC、0.000075%BAC+LY2157299(TGF-PR1抑制劑,200μM、0.000075%BAC+NS-398(COX-2抑制劑,100μ M)作用于細胞,24小時后提取蛋白和RNA。以Western Blot (WB)和qRT-PCR方法檢測結膜組織和原代培養(yǎng)的CFs的細胞外基質(I型膠原a1鏈,CollAl;纖維連接蛋白-1,FN-1),TGF-βp信號通路相關分子(TGF-β1TGF-βR1TGF-βR2 Smad2、 Smad3. P-Smad3)和COX-2的蛋白及mRNA表達水平,并以蘇木素-伊紅染色法(HE)、苦味酸-酸性品紅染色法(Van Gieson's),過碘酸-希夫染色法(PAS)和免疫組織化學染色法觀察結膜下組織的病理改變及炎癥細胞的浸潤情況。 結果：HE、Van Gieson's及PAS染色結果顯示,0.01%BAC處理組球結膜上皮細胞及杯狀細胞的形態(tài)和數(shù)量與PBS對照組相比無明顯不同,但結膜下組織成纖維細胞密度輕微增加,有明顯的膠原纖維沉積；免疫組織化學染色顯示,0.01%BAC處理組球結膜下組織未見中性粒細胞和巨噬細胞浸潤；WB和qRT-PCR結果顯示,BAC處理組球結膜組織和原代培養(yǎng)的CFs CollAl FN-1COX-2, TGF-β1 TGF-βRK Smad3, P-Smad3蛋白或mRNA的表達均顯著高于PBS對照組,而TGF-βpR2和Smad2蛋白的表達與PBS對照組相比無明顯差異；0.000075%BAC+LY2157299細胞處理組,P-Smad3、CollAl和FN-1的蛋白表達均顯著低于0.000075%BAC處理組；0.000075%BAC+NS-398細胞處理組,TGF-β1 TGF-βR1、Smad3、P-Smad3蛋白表達均顯著低于0.000075%BAC處理組。 結論：BAC通過COX-2激活TGF-β1/Smad3信號通路,引起結膜下成纖維細胞產(chǎn)生大量細胞外基質從而引起結膜下組織纖維化。
[Abstract]:Objective: benzalkonium chloride (BAC) is the most commonly used antiseptic in eye drops. A large number of clinical and experimental studies have shown that long-term use of BAC-containing eye drops can cause a series of adverse eye surface reactions. The purpose of this study was to investigate the mechanism of subconjunctival fibrosis induced by BAC. Methods 45 male Sprague-Dawley rats were randomly divided into control group, experimental group and cell culture group with 15 rats in each group. The control group and the experimental group were treated with PBS and 0.01 C drops of left eye twice a day every 12 hours. One month later, protein and RNA were extracted from the conjunctiva of the left eye and paraffin embedded sections were prepared, and CFS of the left conjunctiva fibroblasts of the left eye were taken from the cell culture group for primary culture. PBS, 0.00005C, 0.000075C, 0.000075C, 0.000075C LY2157299TGF-PR1 inhibitor 200 渭 M, 0.000075C NS-398COX-2 inhibitor 100 渭 M) were used to extract protein and RNA for 24 hours. Western blot and qRT-PCR were used to detect the extracellular matrix type I collagen A1 chain collagen A 1 chain of conjunctival tissue and primary cultured CFS. TGF- 尾 1 TGF- 尾 R 1 TGF- 尾 R 2 Smad2, Smad3, TGF- 尾 1 TGF- 尾 R 1 TGF- 尾 R 2, Smad3, TGF- 尾 1 TGF- 尾 R 1 TGF- 尾 R 2, Smad3 were detected by Western blot and qRT-PCR. The protein and mRNA expression levels of P-Smad3) and COX-2, The pathological changes of subconjunctival tissue and the infiltration of inflammatory cells were observed by hematoxylin-eosin staining, picric acid-acid fuchsin staining and periodate-Schiff staining. Results the morphology and number of bulbar conjunctival epithelial cells and goblet cells in 0.01C treated group were not significantly different from those in PBS control group, but the density of fibroblasts in subconjunctival tissue increased slightly. Immunohistochemical staining showed that there was no infiltration of neutrophils and macrophages in subconjunctival tissue of 0.01C group. The results of WB and qRT-PCR showed that the expressions of protein and mRNA in conjunctiva and CFS CollAl FN-1COX-2, TGF- 尾 1TGF- 尾 RK Smad3 and P-Smad3 in BAC treated group were significantly higher than those in PBS control group, but the expression of TGF- 尾 pR2 and Smad2 protein in BAC group was not significantly different from that in PBS control group. The protein expression of P-Smad3A1CollAl and FN-1 in 0.000075C LY2157299 cell group was significantly lower than that in 0.000075C treated group, and the expression of TGF- 尾 1 TGF- 尾 1 TGF- 尾 1 TGF- 尾 Smad3 + P-Smad3 protein in 0.000075C treated group was significantly lower than that in 0.000075C group. ConclusionBAC activates TGF- 尾 1 / Smad3 signaling pathway through COX-2, which results in a large amount of extracellular matrix produced by subconjunctival fibroblasts and subconjunctival fibrosis.
【學位授予單位】：廈門大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R965

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