苯扎氯铵致结膜下组织纤维化机制的研究

发布时间：2018-06-14 00:34

本文选题：苯扎氯铵 + 结膜下组织纤维化　；参考：《厦门大学》2014年硕士论文

【摘要】：目的：苯扎氯铵(benzalkonium chloride, BAC)是滴眼液中最常用的防腐剂。大量临床及实验研究表明,长期使用含BAC的滴眼液会引起一系列的眼表不良反应。本研究主要探讨BAC致结膜下组织纤维化的机制。方法：SD雄性大鼠45只,随机分成对照组、实验组和细胞培养组,每组15只。对照组和实验组分别用PBS和0.01%BAC滴左眼,每天两次,每12小时一次。一个月后,取大鼠左眼球结膜组织提取蛋白和RNA及制备石蜡包埋切片；细胞培养组取大鼠左眼球结膜成纤维细胞(CFs)进行原代培养,分别用PBS、0.00005%BAC、0.000075%BAC、0.000075%BAC+LY2157299(TGF-PR1抑制剂,200μM、0.000075%BAC+NS-398(COX-2抑制剂,100μ M)作用于细胞,24小时后提取蛋白和RNA。以Western Blot (WB)和qRT-PCR方法检测结膜组织和原代培养的CFs的细胞外基质(I型胶原a1链,CollAl;纤维连接蛋白-1,FN-1),TGF-βp信号通路相关分子(TGF-β1TGF-βR1TGF-βR2 Smad2、 Smad3. P-Smad3)和COX-2的蛋白及mRNA表达水平,并以苏木素-伊红染色法(HE)、苦味酸-酸性品红染色法(Van Gieson's),过碘酸-希夫染色法(PAS)和免疫组织化学染色法观察结膜下组织的病理改变及炎症细胞的浸润情况。结果：HE、Van Gieson's及PAS染色结果显示,0.01%BAC处理组球结膜上皮细胞及杯状细胞的形态和数量与PBS对照组相比无明显不同,但结膜下组织成纤维细胞密度轻微增加,有明显的胶原纤维沉积；免疫组织化学染色显示,0.01%BAC处理组球结膜下组织未见中性粒细胞和巨噬细胞浸润；WB和qRT-PCR结果显示,BAC处理组球结膜组织和原代培养的CFs CollAl FN-1COX-2, TGF-β1 TGF-βRK Smad3, P-Smad3蛋白或mRNA的表达均显著高于PBS对照组,而TGF-βpR2和Smad2蛋白的表达与PBS对照组相比无明显差异；0.000075%BAC+LY2157299细胞处理组,P-Smad3、CollAl和FN-1的蛋白表达均显著低于0.000075%BAC处理组；0.000075%BAC+NS-398细胞处理组,TGF-β1 TGF-βR1、Smad3、P-Smad3蛋白表达均显著低于0.000075%BAC处理组。结论：BAC通过COX-2激活TGF-β1/Smad3信号通路,引起结膜下成纤维细胞产生大量细胞外基质从而引起结膜下组织纤维化。
[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.
【学位授予单位】：厦门大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R965

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