脂肪间充质干细胞来源的外泌体对苯扎氯铵诱导的小鼠干眼的治疗作用及机制  

Therapeutic effect and mechanism of exosomes derived from adipose-derived mesenchymal stem cells in benzalkonium chloride induced dry eye in mice

作　　者：王桂芳 龙红梅 龚喜乐缘 胡淑芳[1] 龚灿[1] WANG Guifang;LONG Hongmei;GONG Xileyuan;HU Shufang;GONG Can(Department of Ophthalmology,Loudi Central Hospital,Loudi 417000,Hunan Province,China;Department of Endocrine,Loudi Central Hospital,Loudi 417000,Hunan Province,China)

机构地区：[1]娄底市中心医院眼科,湖南省娄底市417000 [2]娄底市中心医院内分泌科,湖南省娄底市417000

出　　处：《眼科新进展》2021年第10期930-934,共5页Recent Advances in Ophthalmology

基　　金：湖南省自然科学基金青年项目(编号2020JJ5460);湖南省技术创新引导项目(编号2017SK51206);娄底市科学技术局项目(编号娄财教指[2019]61号)。

摘　　要：目的探讨脂肪间充质干细胞来源的外泌体(ADSC-Exos)对苯扎氯铵诱导的小鼠干眼的治疗作用及机制。方法采用超速离心法获取到较高纯度的ADSC-Exos,利用Western blot、纳米粒跟踪分析和透射电镜检测ADSC-Exos特征。取36只小鼠随机分为正常对照组,模型组,低、中、高剂量外泌体组和普拉洛芬组,每组6只。除正常对照组外,其余各组小鼠双眼结膜囊内滴入2 g·L^(-1)苯扎氯铵溶液,每天2次,连续滴眼14 d,制作中重度干眼模型。造模结束后,在正常对照组和模型组小鼠结膜囊内滴入5μL磷酸盐缓冲液,低、中、高剂量外泌体组分别滴12.5 g·L^(-1)、25.0 g·L^(-1)和50.0 g·L^(-1)ADSC-Exos,普拉洛芬组滴相同体积的1 g·L^(-1)普拉洛芬滴眼液,每天3次,连续滴眼7 d。检测各组小鼠角膜荧光素染色评分、酚红棉线湿润长度和泪膜破裂时间。利用流式细胞仪检测各组小鼠结膜组织中白细胞介素(IL)-1α、γ-干扰素(IFN)和肿瘤坏死因子(TNF)-α含量;Western blot和实时荧光定量PCR检测各组小鼠结膜组织中Myd88和Toll样受体4(TLR4)蛋白及mRNA的表达。结果与正常对照组相比,治疗后第4天和第7天,模型组小鼠角膜荧光素染色评分均明显增加,酚红棉线湿润长度和泪膜破裂时间明显缩短(均为P<0.01)。与模型组相比,治疗后第4天,高剂量外泌体组和普拉洛芬组小鼠角膜荧光素染色评分均明显降低(均为P<0.05),酚红棉线湿润长度均明显增加(均为P<0.01),泪膜破裂时间延长,但差异均无统计学意义(均为P>0.05)。与模型组相比,治疗后第7天,中、高剂量外泌体组和普拉洛芬组小鼠角膜荧光素染色评分均显著降低(均为P<0.05),酚红棉线湿润长度均明显增加(均为P<0.01);高剂量外泌体组和普拉洛芬组小鼠泪膜破裂时间均明显延长(均为P<0.01)。与正常对照组相比,模型组小鼠结膜组织中IL^(-1)α、γ-IFN和TNF-α含量明显增加(均为P<0.05)。治疗Objective To investigate the therapeutic effects and mechanisms of adipose-derived mesenchymal stem cell-derived exosomes(ADSC-Exos)on benzalkonium chloride(BAC)induced dry eye in mice.Methods High-purity ADSC-Exos were isolated by ultracentrifugation.Western blot,nanoparticle tracking analysis,and transmission electron microscopy were used to characterize ADSC-Exos.Thirty-six mice were randomly divided into six groups(6 mice per group)normal control group,model group,low-,medium-,and high-dose exosome groups,and pranoprofen group.Except for the normal control group,2 g·L^(-1) BAC was administered to mouse eyes for 14 d,twice daily,to make the moderate-severe dry eye model.After modeling,the normal control group and model group mice were treated with 5μL phosphate buffer solution(PBS);the low-,medium-,and high-dose exosome groups were treated with 12.5 g·L^(-1),25.0 g·L^(-1),and 50.0 g·L^(-1) ADSC-Exos,respectively;and the pranoprofen group was treated with the same dose of 1 g·L^(-1) pranoprofen eye drops for 7 d,t.i.d.Corneal fluorescein staining score,phenol red-impregnated cotton thread,and tear film breakup time were evaluated.The concentrations of interleukin(IL)-1α,interferon-γ(IFN-γ)and tumor necrosis factor(TNF)-αin conjunctival tissues of mice in each group were detected by flow cytometry.The expression levels of MyD88,Toll-like receptor 4(TLR4)protein and mRNA of TLR4 pathway components were assessed by Western blot and qRT-PCR,respectively.Results At 4 d and 7 d after therapy,the corneal fluorescein staining score of model group was higher than that in the normal control group while phenol red-impregnated cotton thread and tear breakup time in the model group were shorter than that in the normal control group(all P<0.01).Compared with the model group,the corneal fluorescein staining score was significantly reduced(both P<0.05),and the phenol red-impregnated cotton thread was significantly increased(borth P<0.01),but the tear film breakup time was extended,no statistical significance(both P>0

关 键 词：脂肪间充质干细胞来源的外泌体 眼表炎症 Toll样受体4 角膜荧光素染色 泪液分泌试验 泪膜破裂时间 

分 类 号：R777.2[医药卫生—眼科]