摘要
背景:在慢性创面愈合过程中,活性氧的过量生成可能会损害L929成纤维细胞的功能,从而延缓创面修复,因此保护成纤维细胞免受氧化应激的影响对于促进创面愈合非常重要。目的:评估羧甲基壳聚糖-氧化硫酸软骨素/富血小板血浆(CMC-OCS/PRP)水凝胶对H2O2刺激下L929细胞的保护作用。方法:制备CMC-OCS/PRP水凝胶,表征水凝胶的微观形貌、降解性能、清除H2O2与羟基自由基的能力及生物相容性。取生长状态良好的L929细胞,分5组培养:对照组常规培养,H2O2组加入H2O2,CMC-OCS组加入羧甲基壳聚糖-氧化硫酸软骨素水凝胶浸提液+H2O2,PRP组加入富血小板血浆凝胶浸提液+H2O2,CMC-OCS/PRP组加入羧甲基壳聚糖-氧化硫酸软骨素/富血小板血浆水凝胶浸提液处理+H2O2,每组先加入水凝胶浸提液处理6 h,然后再加入H2O2处理24 h。培养结束后,检测细胞活性氧及丙二醛水平,细胞凋亡、胶原纤维Ⅰ蛋白表达。在加入H2O2的情况下,将上述水凝胶浸提液分别与L929成纤维细胞直接或间接共培养36 h,通过划痕实验、Transwell小室实验检测细胞的迁移能力。结果与结论:①CMC-OCS/PRP水凝胶具有均匀相互关联的多孔结构及良好的降解能力,体外可有效清除H2O2与羟基自由基,并且具有良好的生物相容性;②与对照组比较,H2O2组细胞凋亡率、细胞活性氧与丙二醛水平升高(P<0.05),细胞铺展面积减少(P<0.05),胶原纤维Ⅰ蛋白表达无明显变化(P>0.05);与H2O2组比较,CMC-OCS组、CMC-OCS/PRP组细胞活性氧水平降低(P<0.05),PRP组、CMC-OCS组、CMCOCS/PRP组丙二醛水平降低(P<0.05)、细胞铺展面积增加(P<0.05),CMC-OCS/PRP组细胞凋亡率降低(P<0.05),PRP组、CMC-OCS组、CMCOCS/PRP组胶原纤维Ⅰ蛋白表达增加(P<0.05);③与对照组比较,H2O2组细胞迁移数量减少(P<0.05),迁移面积无明显变化(P>0.05);与H2O2组比较,PRP组、CMC-OCS组、CMC-OCS/PRP组细胞迁移数量、迁移面积均增加(P<0.05),并且以CMC-OCS/PRP组增加最显著;④在氧化应激条件下,CMC-OCS/PRP水凝胶可改善成纤维细胞的迁移能力,抗细胞凋亡并保护细胞伸展功能。
BACKGROUND:During healing process of chronic wounds,excessive production of reactive oxygen species can impair the function of L929 fibroblasts,thereby delaying wound repair.Therefore,protecting fibroblasts from oxidative stress is important to promote wound healing.OBJECTIVE:To assess the protective effects of carboxymethyl chitosan-oxidized chondroitin sulfate/platelet-rich plasma(CMC-OCS/PRP)hydrogel on L929 cells under H2O2 stimulation.METHODS:CMC-OCS/PRP hydrogels were prepared,and the micromorphology,degradation performance,scavenging ability of H2O2 and hydroxyl radical and biocompatibility of the hydrogels were characterized.L929 cells with good growth state were taken and cultured in five groups.The control group was cultured conventionally.H2O2 was added to the H2O2 group.Carboxymethyl chitosan-oxidized chondroitin sulfate hydrogel extract+H2O2 was added to the CMCOCS group.Platelet-rich plasma gel extract+H2O2 was added to the PRP group.The CMC-OCS/PRP group was treated with carboxymethyl chitosan-oxidized chondroitin sulfate/platelet-rich plasma hydrogel extract+H2O2.Each group was treated with hydrogel extract for 6 hours,and then H2O2 for 24 hours.After culture,the levels of active oxygen and malondialdehyde,apoptosis and expression of collagen fiber I protein were detected.In the presence of H2O2,the above hydrogel extracts were directly or indirectly co-cultured with L929 fibroblasts for 36 hours,respectively.Migration ability of the cells was detected by scratch test and Transwell chamber test.RESULTS AND CONCLUSION:(1)CMC-OCS/PRP hydrogels had uniform and interrelated porous structure and good degradation ability,could effectively remove H2O2 and hydroxyl radicals in vitro,and had good biocompatibility.(2)Compared with the control group,the apoptosis rate,reactive oxygen species,and malondialdehyde levels were increased(P<0.05);the spread area of cells was decreased(P<0.05),and the expression of collagen fiber I protein had no significant changes(P>0.05)in the H2O2 group.Compared with the H2O2 group,reactive oxygen species level was decreased in the CMC-OCS group(P<0.05),malondialdehyde level was decreased(P<0.05),and cell spread area was increased(P<0.05)in the PRP group,CMC-OCS group,and CMC-OCS/PRP group;apoptosis rate was decreased in the CMC-OCS/PRP group(P<0.05),and collagen fiber I protein expression was increased in the PRP group,CMC-OCS group,and CMC-OCS/PRP group(P<0.05).(3)Compared with the control group,the number of cell migration was decreased(P<0.05),and the migration area had no significant change(P>0.05)in the H2O2 group.Compared with the H2O2 group,the number and area of cell migration were increased in the PRP group,CMC-OCS group,and CMC-OCS/PRP group(P<0.05),and the increase was most significant in the CMC-OCS/PRP group.(4)Under oxidative stress,CMC-OCS/PRP hydrogel can improve the migration ability of fibroblasts,resist cell apoptosis,and preserve cell extension function.
作者
王自林
牟秋菊
刘宏杰
申玉雪
祝丽丽
Wang Zilin;Mu Qiuju;Liu Hongjie;Shen Yuxue;Zhu Lili(Teaching and Research Department of Clinical Laboratory Fundamentals and Hematology,School of Medical Laboratory,Guizhou Medical University,Guiyang 550000,Guizhou Province,China;Department of Blood Transfusion,Affiliated Hospital of Guizhou Medical University,Guiyang 550000,Guizhou Province,China)
出处
《中国组织工程研究》
CAS
北大核心
2025年第4期771-779,共9页
Chinese Journal of Tissue Engineering Research
基金
贵州医科大学附属医院高层次人才项目(gyfygcc-2023-03),项目负责人:祝丽丽。
