负载人脐带间充质干细胞来源的小细胞外囊泡的甲基丙烯酸酐化明胶水凝胶治疗小鼠全层皮肤缺损创面的效果  

Effects of gelatin methacrylate anhydride hydrogel loaded with small extracellular vesicles derived from human umbilical cord mesenchymal stem cells in the treatment of full-thickness skin defect wounds in mice

作　　者：陈祎琦 周莹芊 魏茜[1] 谢晓烨 刘馨竹 李大伟[1] 申传安[1] Chen Yiqi;Zhou Yingqian;Wei Qian;Xie Xiaoye;Liu Xinzhu;Li Dawei;Shen Chuan'an(Senior Department of Burns and Plastic Surgery,the Fourth Medical Center of PLA General Hospital,Beijing 100048,China)

机构地区：[1]解放军总医院第四医学中心烧伤整形医学部,北京100048

出　　处：《中华烧伤与创面修复杂志》2024年第4期323-332,共10页Chinese Journal of Burns And Wounds

基　　金：国家自然科学基金面上项目(82272279);北京市自然科学基金青年项目(7244411);解放军总医院第四医学中心自主创新科学基金(2024-4ZX-MS-06)。

摘　　要：目的探究负载人脐带间充质干细胞来源的小细胞外囊泡(hUCMSC-sEV)的甲基丙烯酸酐化明胶(GelMA)水凝胶治疗小鼠全层皮肤缺损创面的效果。方法该研究为实验研究。采用超速离心法提取hUCMSC-sEV,通过透射电子显微镜观察其形貌,采用蛋白质印迹法检测CD9、CD63、肿瘤易感基因101(TSG101)及钙联蛋白的表达。将人脐静脉内皮细胞(HUVEC)及第3、4代人表皮角质形成细胞(HEK)、人真皮成纤维细胞(HDF)均分为常规培养的空白对照组和在细胞培养液中加入hUCMSC-sEV培养的hUCMSC-sEV组,行细胞划痕试验并计算划痕后6、12、24 h的细胞迁移率,行细胞Transwell试验并计算培养12 h细胞迁移数量,行5-乙炔基-2''-脱氧尿嘧啶核苷、Hoechst染色检测培养24 h增殖细胞比例,样本数均为3。制备单纯GelMA水凝胶及负载hUCMSC-sEV的GelMA水凝胶(以下简称hUCMSC-sEV/GelMA水凝胶),通过扫描电子显微镜观察2种水凝胶微观形貌,通过激光扫描共聚焦显微镜观察hUCMSC-sEV的分布情况,采用蛋白质比色定量法测定并计算hUCMSC-sEV/GelMA水凝胶在磷酸盐缓冲液(PBS)中浸泡0(即刻)、2、4、6、8、10、12 d时hUCMSC-sEV累积释放率(样本数为3)。将24只6周龄雄性C57BL/6J小鼠按随机数字表法分为PBS组、单纯hUCMSC-sEV组、单纯GelMA水凝胶组和hUCMSC-sEV/GelMA水凝胶组(每组6只),于小鼠背部制备全层皮肤缺损创面后分别行PBS注射、hUCMSC-sEV悬液注射、单纯GelMA水凝胶覆盖、hUCMSC-sEV/GelMA水凝胶覆盖。于伤后0(即刻)、4、8、12 d观察创面愈合情况并统计伤后4、8、12 d创面愈合率,于伤后12 d取创面组织行苏木精-伊红染色后观察创面新生组织结构,样本数均为6。结果提取的hUCMSC-sEV呈杯状结构,表达CD9、CD63和TSG101,几乎不表达钙联蛋白。划痕后6、12、24 h,hUCMSC-sEV组HEK(t值分别为25.94、20.98、20.04)、HDF(t值分别为3.18、5.68、4.28)、HUVEC(t值分别为4.32、19.33、4.00)的迁�Objective To investigate the effects of gelatin methacrylate anhydride(GelMA)hydrogel loaded with small extracellular vesicles derived from human umbilical cord mesenchymal stem cells(hUCMSCs-sEVs)in the treatment of full-thickness skin defect wounds in mice.Methods This study was an experimental study.hUCMSCs-sEVs were extracted by ultracentrifugation,their morphology was observed through transmission electron microscope,and the expression of CD9,CD63,tumor susceptibility gene 101(TSG101),and calnexin was detected by Western blotting.The human umbilical vein endothelial cells(HUVECs),the 3rd and 4th passages of human epidermal keratinocytes(HEKs)and human dermal fibroblasts(HDFs)were all divided into blank control group(routinely cultured)and hUCMSC-sEV group(cultured with the cell supernatant containing hUCMSCs-sEVs).The cell scratch test was performed and the cell migration rates at 6,12,and 24 h after scratching were calculated,the cell Transwell assay was performed and the number of migration cells at 12 h after culture was calculated,and the proportion of proliferating cells was detected by 5-acetylidene-2'-deoxyuridine and Hoechst staining at 24 h after culture,with sample numbers being all 3.The simple GelMA hydrogel and the GelMA hydrogel loaded with hUCMSCs-sEVs(hereinafter referred to as hUCMSC-sEV/GelMA hydrogel)were prepared.Then the micromorphology of 2 kinds of hydrogels was observed under scanning electron microscope,the distribution of hUCMSCs-sEVs was observed by laser scanning confocal microscope,and the cumulative release rates of hUCMSCs-sEVs at 0(immediately),2,4,6,8,10,and 12 d after soaking hUCMSC-sEV/GelMA hydrogel in phosphate buffer solution(PBS)were measured and calculated by protein colorimetric quantification(n=3).Twenty-four 6-week-old male C57BL/6J mice were divided into PBS group,hUCMSC-sEV alone group,GelMA hydrogel alone group,and hUCMSC-sEV/GelMA hydrogel group according to the random number table,with 6 mice in each group,and after the full-thickness skin defect wounds on the

关 键 词：组织工程 间质干细胞 水凝胶 胞外囊泡 生物相容性材料 

分 类 号：R641[医药卫生—外科学]