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
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)
出处
《山东医药》
CAS
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)。
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
exosome
umbilical cord mesenchymal stem cells
hydrogel
thermoresponsive hydrogel
diabetic chronic refractory cutaneous ulcers
human umbilical vein endothelial cells
cell experiment
