Background Researches showed that transforming growth factor-β2 (TGF-β2) promotes the activity of human Tenon capsular fibroblasts (TFs),which plays a role in the scarring of filtering blebs after antiglaucoma surgery.However,its mechanism is not fully clear.Lysyl oxidases (LOXs) are important extracellular matrix proteases which can catalyze the cross-linking of collagen and elastin.Investigating the impact of TGF-β2 on the expression of LOXs has a great significance for the understanding of the pathogenesis of filtering bleb scarring and its prevention.Objective This study was to investigate the effect of TGF-β2 on the expression of LOXs in cultured human TFs.Methods The TFs at 4-8 generations were divided into normal control group and different concentrations of TGF-β2 treated-groups,and 100,200,400,800 μ1 of TGF-β2 with the final concentration of 2,4,8 and 16 ng/ml was added into the medium to treat human TFs respectively for 24 hours.The LOXs in the cells were detected by Western blot to determine the optimal dose of TGF-β2.The 4 ng/ml TGF-β2(200 μ1) was used to treat human TFs for 6,12,24 and 48 hours respectively,and the change of LOXs expression in the cells over time was assayed by Western blot.The expression and distribution of LOX protein in the normal cells and TGF-β2-treated cells was detected by using immunofluorescence technique.This study was approved by Daping Hospital of Third Military Medical University Ethic Commission.The guardians of the patients who offered the specimen knew the purpose of the study and signed informed consent.Results Western blot assay showed that the expressions of LOX,LOXL1,LOXL2,LOXL3 and LOXL4 in the cells were gradually elevated from the normal control group and 2,4,8,16 ng/ml TGF-β2-treated groups,showing significant differences among the groups (F =37.338,13.438,31.067,11.767,15.167,all at P＜0.01).The expression of LOXL2 protein in the cells was 0.68±0.07,1.09±0.10,1.32±0.07,1.50± 0.06 and 1.89±0.12 in the normal control group and 6-,12-,24-and 48-hour groups respectively after 4 ng/ml TGF-β2 treatment,with a significant increase over time (F =82.832,P=0.000).The expression of LOX was weak in the normal cultured TFs,while the fluorescence intensity of LOX expression was evidently enhanced in the cytoplasm of the cells in the TGF-β2-treated group.Conclusions TGF-β2 upregulates the expressions of LOXs in human TFs in a dose-and time-dependent manner,which probably offers a basis for the further study on the prevention of filtering bleb scarring after glaucoma surgery.

目的:对原始多肽即靶向髓样分化蛋白-2(MD-2)拮抗多肽T6、T11进行突变多肽库的构建、筛选和功能鉴定,以获得具有更高抗炎活性的多肽.方法:以前期合成的可阻断MD-2与内毒素(LPS)结合但效率不高的原始多肽T6和T11序列为基础,每次随机突变相邻的3～5个氨基酸,保留剩余氨基酸,并同时在肽链的氨基端和羧基端引入额外的两个氨基酸,构建大容量的噬菌体突变肽库;再用全长的MD-2蛋白进行靶向淘选获得特异结合的多肽.最后用细胞实验和动物实验验证所获特异多肽的抗炎效果:①用获得的多肽200 μg/ml分别孵育巨噬细胞和人单核白细胞2h,再用内毒素(LPS)1μg/ml刺激6h.以单纯细胞培养液培养的细胞和LPS刺激6h的细胞作对照.②C57小鼠腹腔注射获得的多肽200 μg/ml,1h后腹腔注射LPS 400 μg/ml,6h后取血.以腹腔注射生理盐水1 ml或LPS的小鼠作对照.每组5只小鼠.采用ELISA法测定各组细胞培养上清和各组小鼠血清的肿瘤坏死因子α(TNF-α)和白介素6(IL-6)的表达水平.结果:成功构建出原始多肽T6和T11序列为基础的突变肽库,用全长的MD-2蛋白进行靶向淘选获得特异结合多肽A8、H2、F5、H5、H9,通过细胞实验筛选得到有抑炎效果的多肽2条(A8、H2),可以明显抑制LPS所致细胞分泌TNF-α和IL-6水平的增高.动物实验发现只有1条多肽(H2)可以明显抑制LPS所致小鼠血清TNF-α和IL-6水平的增高.结论:成功构建了突变肽库,并从中筛选出一条具有高效抗炎效应的多肽.

Objective The present study investigates the impact of fibronectin(Fn) on the inflammatory factor release of human peripheral blood mononuclear cells(PBMCs) and TLR4 expression.Methods Gradient centrifugation was used to separate normal PBMCs from concentrated human PBMCs.The blank control,Fn stimulus,LPS stimulus,and Fn plus LPS complex stimulus groups were established to stimulate the PBMCs for 0.5,2,4,8,and 12 h,respectively.Subsequently,cell culture supernatant samples were collected to test the inflammatory factors IL-6,IL-10,and TNF-α using ELISAs,and Western blot was used to detect the effect of every stimulus on Toll-like receptor 4(TLR4) expression.Results At the 4 and 8 h time points,the IL-6 production in the Fn plus LPS group reached 727±37.73 and 918.25±26.78 pg/ml,respectively,which were much higher than those in the other groups(P < 0.05).Moreover,the TNF-α production in the Fn plus LPS group increased 2 h after stimulation and was significantly higher than those in the other three groups(P < 0.05).The IL-10 productions in the Fn plus LPS group showed a slowly increasing trend at the 0.5,2,and 4 h time points and reached 784.74±91.96 and 1081.74±57.98 pg/ml at the 8 and 12 h time points,respectively.Again,these results were significantly higher than those in the other groups(P < 0.05).The Western blot analysis showed that the expression level of TLR4 in the complex stimulus group was evidently higher than those in the other three groups at each time point(P < 0.05).Conclusion Fn or LPS stimulation can therefore promote the expression of TLR4 in PBMCs.However,their combination can remarkably increase the expression of TLR4 and the production of IL-6 and TNF-α.These results imply that Fn can enhance the sensitivity of PBMCs to LPS.