摘要
目的探讨间充质干细胞(MSCs)负载转铁蛋白(Tf)对实验性青光眼大鼠的保护作用及其分子机制。方法2023年3月至2024年6月间40只SD大鼠按照随机数字表法分为对照组、模型组、MSCs组和MSCs-Tf组,每组10只,模型组、MSCs组和MSCs-Tf组大鼠采用激光光凝大鼠房角诱导高眼压,持续1周建立青光眼大鼠模型,对照组大鼠不处理。建模成功后,MSCs组和MSCs-Tf组大鼠经玻璃体腔注射对照组和模型大鼠MSCs或MSCs-Tf(1×106个/眼)。模型组和对照组大鼠注射等体积生理盐水。连续干预7 d,4周后进行观察。采用免疫检测MSCs在视网膜的定植;采用眼压仪测定4组大鼠眼压变化;采用荧光金标记法检测4组大鼠视网膜神经节细胞数量;苏木精-伊红(HE)染色分析4组大鼠视网膜内丛状层、内核层、外核层厚度;采用试剂盒检测4组大鼠测视网膜超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)水平;采用酶联免疫吸附试验(ELISA)试剂盒测定炎性因子水平;采用蛋白质免疫印迹检测转铁蛋白受体(TfR)和铁蛋白(Ferritin)表达。组间计量数据比较采用单因素方差分析。结果MSCs组和MSCs-Tf组大鼠视网膜组织中MSCs定植率[(47.91±12.43)%、(52.18±8.73)%]明显高于模型组[(0.00±0.00)%],差异有统计学意义(t=12.150、15.361,P<0.05)。MSCs-Tf组大鼠视网膜内丛状层和内核层厚度[(46.57±5.82)、(27.38±4.14)μm]明显高于MSCs组[(32.77±4.02)、(22.47±2.29)μm],差异有统计学意义(t=6.173、3.275,P<0.05)。MSCs-Tf组视网膜神经节细胞比例[(51.78±7.11)%]明显高于MSCs组[(37.28±4.01)%],差异有统计学意义(t=5.617,P<0.05)。MSCs-Tf组大鼠视网膜组织SOD和GSH-Px活性[(71.74±6.08)、(615.44±74.61)U/L]明显高于MSCs组[(56.27±5.37)、(459.45±63.78)U/L],差异有统计学意义(t=6.029、5.026,P<0.05)。MSCs-Tf组大鼠视网膜肿瘤坏死因子-α(TNF-α)和IL-1β水平[(63.26±7.83)、(117.76±25.88)pmol/L]明显低于MSCs组[(102.48±9.23)、(253.60±34.41)pmol/L],差异有统计学意义(t=10.250、9.977,P<0.05)。MSCs-Tf组大鼠视网膜组织转铁蛋白受体和转铁蛋白表达水平(0.86±0.06、1.71±0.17)明显高于MSCs组大鼠(0.62±0.08、0.79±0.07),差异有统计学意义(t=8.161、16.110,P<0.04)。结论MSCs负载转铁蛋白提高视网膜神经节细胞存活率,减少视神经萎缩,主要通过调节铁代谢和抗氧化途径发挥协同保护作用。
Objective To investigate the protective effect and molecular mechanisms of transferrin(Tf)-loaded mesenchymal stem cells(MSCs)on glaucoma in rats.Methods Totally,40 Sprague-Dawley(SD)rats were randomly divided into 4 groups(n=10 per group):control group,model group,MSCs group,and MSCs-Tf group from March 2023 to June 2024.Glaucoma was induced in the model,MSCs,and MSCs-Tf groups by laser photocoagulation of the anterior chamber angle to elevate intraocular pressure(IOP)for one week,while the control group remained untreated.After successful modeling,the MSCs and MSCs-Tf groups received intravitreal injections of MSCs or MSCs-Tf(1×106 cells/eye),respectively,whereas the model and control groups received an equal volume of saline.Interventions lasted for 7 days,and observations were conducted at 4th week.MSC engraftment in the retina was assessed via immunofluorescence.IOP changes were measured using a tonometer.Retinal ganglion cell(RGC)survival was evaluated via FluoroGold labeling.Hematoxylin-eosin(HE)staining was used to analyze the thickness of the inner plexiform layer(IPL),inner nuclear layer(INL),and outer nuclear layer(ONL).Superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)levels were measured using assay kits.Inflammatory cytokine levels were quantified via enzyme linked immunosorbent assay(ELISA).Transferrin receptor(TfR)and ferritin expression were analyzed by Western blotting.Intergroup comparisons were analyzed using one-way ANOVA.Results MSC engraftment rates in the MSCs and MSCs-Tf groups[(47.91±12.43)%,(52.18±8.73)%]were significantly higher than in the model group[(0.00±0.00)%,t=12.150,15.361,P<0.05].The IPL and INL thicknesses in the MSCs-Tf group[(46.57±5.82),(27.38±4.14)μm]were significantly greater than those in the MSCs group[(32.77±4.02),(22.47±2.29)μm,t=6.173,3.275,P<0.05].The RGC survival rate in the MSCs-Tf group[(51.78±7.11)%]was significantly higher than in the MSCs group[(37.28±4.01)%,t=5.617,P<0.05].Retinal SOD and GSH-Px activities in the MSCs-Tf group[(71.74±6.08),(615.44±74.61)U/L]were significantly elevated compared to the MSCs group[(56.27±5.37),(459.45±63.78)U/L,t=6.029,5.026,P<0.05].Tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)levels in the MSCs-Tf group[(63.26±7.83),(117.76±25.88)pmol/L]were significantly lower than in the MSCs group[(102.48±9.23),(253.60±34.41)pmol/L,t=10.250,9.977,P<0.05].TfR and ferritin expression levels in the MSCs-Tf group(0.86±0.06,1.71±0.17)were significantly higher than in the MSCs group(0.62±0.08,0.79±0.07,t=8.161,16.110,P<0.05).Conclusion Transferrin-loaded MSCs enhance RGC survival and mitigate optic nerve atrophy,primarily through synergistic modulation of iron metabolism and antioxidative pathways.
作者
杨华
曹慧
张丽阳
李新民
朱绍辉
Yang Hua;Cao Hui;Zhang Liyang;Li Xinmin;Zhu Shaohui(Department Of Ophthalmology,the First Affiliated Hospital of Xinxiang Medical University,Weihui 453100,China;Department of General Surgery,the First Affiliated Hospital of Xinxiang Medical University,Weihui 453100,China)
出处
《中华实验外科杂志》
2025年第7期1332-1336,共5页
Chinese Journal of Experimental Surgery
基金
2022年度河南省医学科技攻关计划联合共建项目(LHGJ20230515)。
