脐带间充质干细胞来源的外泌体-温敏水凝胶的构建及侵入性观察  

Construction of exosome-temperature-sensitive hydrogel derived from umbilical cord mesenchymal stem cells and its invasiveness

作　　者：陈智毅 潘道延 杨嘉旖 林凯桑 胡世弟 张彤 周凌燕 沈洁 CHEN Zhiyi;PAN Daoyan;YANG Jiayi;LIN Kaisang;HU Shidi;ZHANG Tong;ZHOU Lingyan;SHEN Jie(The Third Affiliated Hospital of Southern Medical University,Guangzhou 510000,China)

机构地区：[1]南方医科大学第三附属医院,广州510000

出　　处：《山东医药》2020年第9期53-56,共4页Shandong Medical Journal

基　　金：广州市健康医疗协同创新重大专项(201604020007)。

摘　　要：目的构建脐带间充质干细胞来源的外泌体-温敏水凝胶,观察其温敏性和侵入性。方法取脐带间充质干细胞株Saliai-HMSC(UC)-N,采用沉淀法提取外泌体,PBS重悬后获得脐带间充质干细胞来源的外泌体重悬液。取Pluronic F-127粉末,避光条件下按20%、22%、24%、26%、28%的浓度梯度分别加入脐带间充质干细胞来源的外泌体重悬液,吹打、静置后,即得到20%、22%、24%、26%、28%浓度的外泌体-温敏水凝胶溶液。将不同浓度的外泌体-温敏水凝胶溶液加入恒温水浴锅,测定初始凝胶化温度、37℃初始凝胶时间。使用PKH67对外泌体进行标记,配置PKH67标记的脐带间充质干细胞来源的外泌体重悬液和PKH67标记的24%浓度的外泌体-温敏水凝胶溶液。取脐静脉内皮细胞分成外泌体组、外泌体-温敏水凝胶组。外泌体组加入PKH67标记的脐带间充质干细胞来源的外泌体重悬液,外泌体-温敏水凝胶组加入PKH67标记的24%浓度的外泌体-温敏水凝胶溶液。两组细胞避光孵育12 h后,滴加稀释的DAPI细胞核染色液,避光孵育30 min,置于荧光倒置显微镜下观察细胞内绿色荧光(绿色荧光为PKH67标记的外泌体)分布情况,并使用Image J软件计算绿色荧光光密度百分比、绿色荧光面积百分比。结果20%、22%、24%、26%、28%浓度的外泌体-温敏水凝胶溶液的初始凝胶化温度分别为(17.8±0.7)、(16.6±0.3)、(14.6±0.5)、(13.4±0.3)、(12.4±0.1)℃,37℃初始凝胶时间分别为(92±6)、(79±5)、(68±3)、(51±3)、(46±2)s,各浓度外泌体-温敏水凝胶溶液的初始凝胶化温度、37℃初始凝胶时间相比,P均<0.05,表明随着温度的变化,外泌体-温敏水凝胶的温敏特性随之变化,而24%浓度的外泌体-温敏水凝胶溶液应用更为简便。外泌体组、外泌体-温敏水凝胶组绿色荧光点分布在细胞质内,并于细胞核周围富集。外泌体组绿色荧光光密度百分比、绿色荧光面积百分比分�Objective We constructed an exosome-temperature-sensitive hydrogel derived from umbilical cord mesenchymal stem cells and observed its temperature sensitivity and invasiveness.Methods Umbilical cord mesenchymal stem cell line Saliai-HMSC(UC)-N-derived exosomes were extracted by using a precipitation method,and then were resuspended in PBS.Under dark conditions,Pluronic F-127 with 20%,22%,24%,26%,and 28%concentration gradients was added to the exosome suspension to obtain 20%,22%,24%,26%,28%concentration exosomes-temperature-sensitive hydrogel solution.Subsequently,the initial gelation temperature and initial gelation time of 37℃were measured for hydrogel solutions of different concentrations.PKH67 was used to label exosomes,and the labeled exosome suspension and exosome-temperature-sensitive hydrogel solution were configured.Umbilical vein endothelial cells were divided into the exosomes group and exosomes-temperature-sensitive hydrogel group;the exosomes group was added with PKH67-labeled exo-some suspension,and the exosomes-temperature-sensitive hydrogel group was added with PKH67-labeled exosomes-temperature-sensitive hydrogel solution at 24%concentration.After 12-hour incubation in the dark,the nucleus was stained with DAPI,and the distribution of green fluorescence in the cells was observed under a fluorescent inverted microscope.The percentage of green fluorescence optical density and green fluorescence area were calculated by using Image J software.Results The initial gelation temperatures of 20%,22%,24%,26%,and 28%exosomes-temperature-sensitive hydrogel solutions were(17.8±0.7),(16.6±0.3),(14.6±0.5),(13.4±0.3),and(12.4±0.1)℃,and 37℃initial gel time is(92±6),(79±5),(68±3),(51±3),and(46±2)s(all P<0.05).It showed that the temperature-sensitive characteristics of exosomes-temperature-sensitive hydrogels changed with temperature fluctuations,and the application of 24%concentration hydrogels had more advantages.The green fluorescent spots in the exosomes group and exosome-temperature-sensitive h

关 键 词：外泌体 脐带间充质干细胞 水凝胶 温敏水凝胶 糖尿病慢性难愈性溃疡 脐静脉内皮细胞 细胞实验 

分 类 号：R587.2[医药卫生—内分泌]