骨髓间充质干细胞来源的外泌体对缺氧/复氧诱导的肾小管上皮细胞损伤的影响  

Protective effect of bone marrow mesenchymal stem cell-derived exosomes on HK-2 cells with hypoxia-reoxygenation injury through regulating Beclin1 expression

作　　者：洪梦琪 俞棋严 李新科 赵宇 HONG Mengqi;YU Qiyan;LI Xinke;ZHAO Yu(Ningbo Ninth Hospital,Ningbo 315020,Zhejiang,China)

机构地区：[1]宁波市第九医院,宁波315020 [2]宁波市鄞州区第二医院

出　　处：《现代实用医学》2024年第7期854-859,共6页Modern Practical Medicine

基　　金：宁波市卫生健康科技计划项目(2023Y40);宁波市鄞州区农业和社会发展项目(2021AS0063)。

摘　　要：目的探讨骨髓间充质干细胞来源外泌体(BMSCs-Exo)对缺氧/复氧(H/R)诱导的肾小管上皮细胞(HK-2)损伤的保护作用及其分子机制。方法外泌体试剂盒提取BMSCs-Exo,透射电镜观察其形态,Western blot检测外泌体表面特异性标志蛋白,采用PHK67绿色荧光标记外泌体并观察HK-2细胞摄取现象。将HK-2细胞分为对照组(NC)、正常组+外泌体组(Exo)、H/R诱导组(H/R)、H/R+外泌体组(H/R+Exo),采用CCK-8法检测细胞活力,流式细胞仪检测细胞凋亡,活性氧(ROS)荧光法测试盒检测ROS水平,氧化应激相关检测试剂盒测定丙二醛(MDA)、谷胱甘肽(GSH)含量。qRT-PCR、Western blot及免疫荧光法检测细胞自噬标志蛋白Beclin1的表达水平。结果成功获得BMSCs-Exo并观察到它可被HK-2细胞摄取。与NC组相比,H/R组HK-2细胞活力、GSH水平显著降低,而凋亡率、ROS、MDA水平显著升高(均P<0.05);与H/R组相比,H/R+Exo组细胞活力、GSH水平则显著增强,凋亡率、ROS、MDA水平下降(均P<0.05);与NC组相比,H/R组Beclin1表达升高,在H/R组加入外泌体后,H/R+Exo组Beclin1表达显著降低(均P<0.05)。结论BMSCs-Exo能通过上调Beclin1改善H/R诱导的HK-2的损伤。Objective To investigate the protective effect and molecular mechanism of bone marrow mesenchymal stem cells(BMSCs)-derived exosomes against hypoxia-reoxygenation(H/R)injury in human tubular epithelial cells(HK-2).Methods BMSCs Exo were extracted using an extracellular vesicle kit,and their morphology was observed under transmission electron microscopy.Western blot was used to detect specific marker proteins on the surface of the extracellular vesicles.PHK67 green fluorescence was used to label the extracellular vesicles and observe the uptake phenomenon of HK-2 cells.HK-2 cells were divided into control group(NC),normal group+exosome group(Exo),H/R induction group(H/R),and H/R+exosome group(H/R+Exo).CCK-8 assay was used to detect cell viability;flow cytometry was used to detect cell apoptosisrate;immunofluorescence staining was used to detect reactive oxygen species(ROS)level;oxidative stress-related detection kits were used to determine MDA,GSH content;quantitative real time polymerase chain reaction(qRT-PCR),Western blot,immunofluorescence staining were used to detect the expression level of Beclin1 in the HK-2 cells.Results BMSCs Exo was successfully obtained and could be taken up by HK-2 cells.Compared with NC group,cell viability and GSH level in H/R group significantly decreased,while cell apoptosis,ROS,MDA significantly improved(all<0.05).Compared with H/R group,cell viability,GSH level group were significantly improved and cell apoptosis,ROS,MDA were significantly decreased in H/R+E xo group(all<0.05).Compared with NC group,the expression level of Beclin1 in H/R group wassignificantly improved,after treatment with exosomes in H/R group,the expression level of Beclin1 significantly decreased in H/R+Exo group(all<0.05).Conclusions BMSCs-Exo can alleviate H/R-induced injury of HK-2 cells by downregu lating the expression of Beclin1.

关 键 词：外泌体 肾小管上皮细胞 缺氧/复氧 BECLIN1 

分 类 号：R692[医药卫生—泌尿科学]