人脂肪间充质干细胞来源细胞外囊泡对高糖诱导的人脐静脉内皮细胞焦亡的影响及其机制  

Influence and mechanism of extracellular vesicles derived from human adipose-derived mesenchymal stem cells on pyroptosis of human umbilical vein endothelial cells induced by high glucose

作　　者：冯俊云 费潇 方邵一涵 安靖雯 施彦 刘德伍[1] Feng Junyun;Fei Xiao;Fang Shaoyihan;An Jingwen;Shi Yan;Liu Dewu(Medical Center of Burn Plastic and Wound Repair,the First Affiliated Hospital,Jiangxi Medical College,Nanchang University,Nanchang 330006,China;Medical College of Nanchang University,Nanchang 330006,China)

机构地区：[1]南昌大学第一附属医院烧伤整形与创面修复医学中心,南昌330006 [2]南昌大学医学院,南昌330006

出　　处：《中华烧伤与创面修复杂志》2025年第3期258-267,共10页Chinese Journal of Burns And Wounds

基　　金：国家自然科学基金地区科学基金项目(82460447,81460293);中医药防治代谢性疾病传承创新团队(2023090006KJZX2022WJW008)。

摘　　要：目的探讨人脂肪间充质干细胞(hADMSC)来源细胞外囊泡(EV),即hADMSC-EV对高糖诱导的人脐静脉内皮细胞(HUVEC)焦亡的影响及其机制,为改善糖尿病创面中血管功能提供依据。方法该研究为实验研究。收集2023年6—9月于南昌大学第一附属医院妇产科完成正常阴道分娩的5名25~40岁产妇的脐带,分离HUVEC并成功鉴定;取同期于该院整形外科行腹部抽脂术的6名25~35岁健康女性的脂肪组织,分离hADMSC后提取hADMSC-EV并成功鉴定。将用含物质的量浓度为33 mmol/L葡萄糖的内皮细胞培养基培养的第4代HUVEC,分为加入磷酸盐缓冲液(PBS)培养的PBS组、加入hADMSC-EV培养的EV组、加入磷脂酰肌醇3-激酶/蛋白激酶B(PI3K/Akt)信号通路抑制剂LY294002和hADMSC-EV培养的EV+LY294002组,采用蛋白质印迹法检测细胞培养48 h后PI3K/Akt信号通路相关蛋白PI3K、Akt和焦亡相关蛋白核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)、胱天蛋白酶1(caspase-1)、消皮素D、白细胞介素1β(IL-1β)、IL-18的蛋白表达;采用细胞计数试剂盒-8检测细胞培养0(即刻)、12、24、36、48、60、72 h增殖水平;培养48 h后,行细胞划痕试验并计算划痕后12、24 h的细胞迁移率,行细胞Transwell试验并计算细胞24 h迁移数量,行细胞成管实验并测算细胞成管总长度与分支节点数。样本数均为3。结果培养48 h后,EV组细胞中PI3K和Akt的蛋白表达均明显高于PBS组(P<0.05),EV+LY294002组细胞中PI3K和Akt的蛋白表达均明显低于EV组(P<0.05)。培养48 h后,EV组细胞中NLRP3、caspase-1、消皮素D、IL-1β、IL-18的蛋白表达分别为0.54±0.08、0.96±0.11、0.525±0.061、1.216±0.039、1.317±0.023,均明显低于PBS组的2.32±0.11、1.86±0.07、1.256±0.113、2.589±0.084、2.042±0.132(P<0.05);EV+LY294002组细胞中NLRP3、caspase-1、消皮素D、IL-1β、IL-18的蛋白表达分别为1.16±0.05、1.37±0.06、0.962±0.028、1.834±0.017、1.803±0.065,均明显高于EV组(P<0.ObjectiveTo investigate the influence and mechanism of extracellular vesicles(EVs)derived from human adipose-derived mesenchymal stem cells(hADMSCs),i.e.hADMSC-EVs on pyroptosis of human umbilical vein endothelial cells(HUVECs)induced by high glucose,with the aim of providing evidence for improving vascular function in diabetic wounds.MethodsThis study was an experimental research.The umbilical cords from 5 women aged 25 to 40 years were collected who had normal vaginal delivery at the Department of Obstetrics and Gynecology of the First Affiliated Hospital of Nanchang University from June to September in 2023,and HUVECs were isolated and successfully identified.Adipose tissue was obtained from 6 healthy women aged 25 to 35 years who underwent abdomen liposuction at the Department of Plastic Surgery of the above-mentioned hospital in the same period.After hADMSCs were isolated,hADMSC-EVs were extracted and successfully identified.The fourth passage of HUVECs were cultured in endothelial cell medium containing glucose in a molarity of 33 mmol/L and divided into phosphate buffered solution(PBS)group cultured with PBS,EV group cultured with hADMSC-EVs,and EV+LY294002 group cultured with hADMSC-EVs and the phosphatidylinositol 3-kinase/protein kinase B(PI3K/Akt)signaling pathway inhibitor LY294002.Western blotting was used to detect the expressions of PI3K/Akt signaling pathway-related proteins PI3K and Akt,and pyroptosis-related proteins nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3),cysteinyl aspartate specific protease-1(caspase-1),gasdermin D,interleukin-1β(IL-1β),and IL-18 of cells after 48 hours of culture.A cell counting kit-8 was used to test the proliferation levels of cells at 0(immediately),12,24,36,48,60,and 72 hours of culture.After 48 hours of culture,the cell scratch test was performed and the cell migration rates at 12 and 24 hours after scratching were calculated;the cell Transwell assay was conducted and the number of cells migrating in 24 hours was calculated;the cell tu

关 键 词：糖尿病 胞外囊泡 人脐静脉内皮细胞 细胞焦亡 磷酸肌醇3-激酶 脂肪间充质干细胞 蛋白激酶B 

分 类 号：R587.1[医药卫生—内分泌]