羧甲基壳聚糖及羧甲基甲壳素对TGF-β诱导的角膜上皮纤维化的抑制作用研究
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摘要
本实验旨在研究不同分子量的羧甲基壳聚糖(CMCTS)和羧甲基甲壳素(CMCT)对转化生长因子β(TGF-β)诱导的人角膜上皮细胞纤维化的抑制作用,并初探其作用机制。MTT法检测不同分子量的CMCTS和CMCT对人角膜上皮细胞系(HCE)生长增殖能力的影响;细胞划痕实验研究TGF-β诱导的HCE细胞纤维化过程中CMCTS和CMCT的作用;western blot方法在蛋白水平探究不同分子量的CMCTS和CMCT对角膜上皮细胞纤维化相关的TGF-β/Smad通路信号分子的影响。结果表明,在10~1 000μg/mL浓度范围内,4种多糖对HCE细胞均具有较好的相容性。此外,不论大分子还是小分子的CMCTS对TGF-β诱导的HCE细胞纤维化均具有显著的抑制作用,其作用机制与抑制TGF-β/Smad信号通路Smad2和Smad3分子的磷酸化有关。然而,两种分子量的CMCT均没有抑制纤维化的作用。因此,CMCTS可以作为一种有发展潜力的生物医用材料应用于人角膜上皮损伤修复,发挥其抗纤维化的作用。
In this study,carboxymethyl chitosan(CMCTS)and carboxymethyl chitin(CMCT)with different molecular weights were explored on their anti-fibrosis effect and the affecting mechanism.The effects of different kinds of CMCTS and CMCT on the growth of the human corneal epithelial cell line(HCE)were measured by MTT assay,while their anti-fibrosis effects on the migration of HCE cells were reflected by wound healing assay.The anti-fibrosis mechanism of different kinds of CMCTS and CMCT in corneal epithelia reconstruction was further studied on protein level.Results showed that CMCTS and CMCT with different molecular weights all suited the proliferation of HCE cells within the concentration of 10~1 000μg/mL.But the two kinds of CMCTS could exert inhibitory effect on the fibrosis of HCE cells induced by TGF-β,and the anti-fibrosis mechanism was related to reducing phosphorylation of Smad2 and Smad3protein in the TGF-β/Smad signaling pathway.Therefore,the two kinds of CMCTS could be applied in the repair of human corneal epithelial injury,playing an important role in anti-fibrosis.
In this study,carboxymethyl chitosan(CMCTS)and carboxymethyl chitin(CMCT)with different molecular weights were explored on their anti-fibrosis effect and the affecting mechanism.The effects of different kinds of CMCTS and CMCT on the growth of the human corneal epithelial cell line(HCE)were measured by MTT assay,while their anti-fibrosis effects on the migration of HCE cells were reflected by wound healing assay.The anti-fibrosis mechanism of different kinds of CMCTS and CMCT in corneal epithelia reconstruction was further studied on protein level.Results showed that CMCTS and CMCT with different molecular weights all suited the proliferation of HCE cells within the concentration of 10~1 000μg/mL.But the two kinds of CMCTS could exert inhibitory effect on the fibrosis of HCE cells induced by TGF-β,and the anti-fibrosis mechanism was related to reducing phosphorylation of Smad2 and Smad3protein in the TGF-β/Smad signaling pathway.Therefore,the two kinds of CMCTS could be applied in the repair of human corneal epithelial injury,playing an important role in anti-fibrosis.
引文
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[3]高美玲,汪东风,杨伟,等.离子交联壳聚糖/海藻酸钠可降解复合膜的研究[J].中国海洋大学学报(自然科学版),2011,41(10):61-66.Gao M L,Wang D F,Yang W,et al.Ionic cross-linking chitosan/alginate biodegradable composite film[J].Periodical of Ocean University of China,2011,41(10):61-66.
[4]王慧,宋福来,刘万顺,等.壳聚糖基水凝胶对肿瘤在体内外生长的影响研究[J].中国海洋大学学报(自然科学版),2015,45(3):87-91.Wang H,Song F L,Liu W S,et al.Effects of a chitosan-based hydrogel on tumor growth in vitro and in vivo study[J].Periodical of Ocean University of China,2015,45(3):87-91.
[5]易喻,江威,王鸿,等.羧甲基壳聚糖的制备及性能研究[J].浙江工业大学学报,2011,39(1):16-20.Yi Y,Jiang W,Wang H,et al.Study on preparation and the performance of the carboxymethyl chitosan[J].Journal of Zhejiang University of Technology,2011,39(1):16-20.
[6]刘万顺,郑美玲,韩宝芹,等.羧甲基壳聚糖对肿瘤生长的影响研究[J].中国海洋大学学报(自然科学版),2011,41(9):36-40.Liu W S,Zheng M L,Han B Q,et al.Study on the antitumor effect of carboxymethyl chitosan[J].Periodical of Ocean University of China,2011,41(9):36-40.
[7]Torricelli A A,Santhanam A,Wu J,et al.The corneal fibrosis response to epithelial-stromal injury[J].Experimental eye research,2016,142:110-118.
[8]Chen Q,Lu H,Yang H.Chitosan inhibits fibroblasts growth in Achilles tendon via TGF-beta1/Smad3pathway by miR-29b[J].International Journal of Clinical and Experimental Pathology,2014,7(12):8462-8470.
[9]Chang J,Liu W,Han B,et al.Investigation of the skin repair and healing mechanism of N-carboxymethyl chitosan in second-degree burn wounds[J].Wound Repair and Regeneration,2013,21(1):113-121.
[10]Wu W,Lee S Y,Wu X,et al.Neuroprotective ferulic acid(FA)-glycol chitosan(GC)nanoparticles for functional restoration of traumatically injured spinal cord[J].Biomaterials,2014,35(7):2355-2364.
[11]Wang D,Mo J,Pan S,et al.Prevention of postoperative peritoneal adhesions by O-carboxymethyl chitosan in a rat cecal abrasion model[J].Clinical and Investigative Medicine Medecine Clinique et Experimentale,2010,33(4):254-260.
[12]Finnson K W,McLean S,Di Guglielmo G M,et al.Dynamics of transforming growth factor beta signaling in wound healing and scarring[J].Advances in Wound Care,2013,2(5):195-214.
[13]Tandon A,Tovey J C,Sharma A,et al.Role of transforming growth factor Beta in corneal function,biology and pathology[J].Current Molecular Medicine,2010,10(6):565-578.
[14]Xu F,Liu C,Zhou D,et al.TGF-beta/SMAD Pathway and its regulation in hepatic fibrosis[J].The Journal of Histochemistry and Cytochemistry:Official Journal of the Histochemistry Society,2016,64(3):157-167.
[15]崔娟娟,冯占芹,张守强,等.羧甲基壳聚糖冲洗液预防大鼠术后腹膜粘连[J].中国组织工程研究,2014(8):1250-1256.Cui J J,Feng Z Q,Zhang S Q,et al.Preventive effect of carboxymethyl chitosan solution on postoperative peritoneal adhesions in a rat model[J].Journal of Clinical Rehabilitative Tissue Engineering Research,2014(8):1250-1256.
[16]Huang X,Zhang Y,Zhang X,et al.Influence of radiation crosslinked carboxymethyl-chitosan/gelatin hydrogel on cutaneous wound healing[J].Materials Science&Engineering C,Materials for Biological Applications,2013,33(8):4816-4824.
[17]Ren C,Zhao D,Zhu L.Use of N,O-carboxymethyl chitosan to prevent postsurgical adhesions in a rabbit double uterine horn model:a randomized controlled design[J].Science China Life Sciences,2016,59(5):504-509.
[18]Saika S,Yamanaka O,Sumioka T,et al.Transforming growth factor beta signal transduction:apotential target for maintenance/restoration of transparency of the cornea[J].Eye&Contact Lens,2010,36(5):286-289.
[19]付玲珠,郑婷,张永生.TGF-β/Smad信号转导通路与肝纤维化研究进展[J].中国临床药理学与治疗学,2014,19(10):1189-1195.Fu L Z,Zheng T,Zhanf Y S,et al.Advances in understanding the role of TGF-β/Smad signalling pathways in the pathogenesis of liver fibrosis[J].Chinese Journal of Clinical Pharmacology and Therapeutics,2014,19(10):1189-1195.
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