人脂肪源细胞外囊泡联合脱细胞脂肪组织支架构建组织工程脂肪  

Construction of tissue engineered adipose by human adipose tissue derived extracellular vesicle combined with decellularized adipose tissues scaffold

作　　者：聂佳莹 易阳艳[1] 朱元正[1] NIE Jiaying;YI Yangyan;ZHU Vuanzheng(Department of Plastic Surgery,the Second Affiliated Hospital of Nanchang University,Nanchang Jiangxi,330006,P.R.China)

机构地区：[1]南昌大学第二附属医院整形美容科

出　　处：《中国修复重建外科杂志》2020年第2期226-233,共8页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(81660326);江西省自然科学基金资助项目(20171ACB20037);江西省卫生厅课题资助项目(20204355)~~

摘　　要：目的探讨人脂肪源细胞外囊泡(human adipose tissue derived extracellular vesicle,hAT-EV)联合脱细胞脂肪组织支架(decellularized adipose tissues,DAT)构建组织工程脂肪的可能性,为临床上软组织缺损修复提供新方案。方法将吸脂手术患者自愿捐赠的脂肪组织分为2份,1份脂肪组织进行脱细胞处理,并采用组织学(HE、Masson染色)、扫描电镜观察,Ⅰ、Ⅳ型胶原及层粘连蛋白免疫组织化学染色和Western blot检测进行表征。另1份脂肪组织使用去外泌体的完全培养基孵化48 h,离心收集培养基上清并使用超高速离心法获取hATEV。使用透射电镜观察其形态,纳米颗粒跟踪分析仪NanoSight分析hAT-EV粒径分布范围,Western blot检测分析hAT-EV膜表面蛋白。将PKH26荧光标记的hAT-EV与脂肪干细胞(adipose derived stem cells,ADSCs)共培养后,共聚焦荧光显微镜观察hAT-EV能否进入ADSCs;将hAT-EV与ADSCs共培养15 d,油红O染色评估其成脂效果。将DAT组织剪碎并注射至8只6周龄雌性C57小鼠背部两侧,左侧每周注射0.2 mL hAT-EV作为实验组,右侧每周注射0.2 mL PBS作为对照组。12周后处死小鼠,取两侧DAT新生物进行湿重称量,并通过HE染色和围脂滴蛋白免疫荧光染色评估hAT-EV在体内诱导脂肪新生的能力。结果脂肪组织经脱细胞后,HE、Masson染色示DAT主要由排列松散的胶原构成,未见细胞核;扫描电镜示DAT中未发现细胞和细胞碎片,同时可见到粗大的胶原纤维束;免疫组织化学染色及Western blot检测示,DAT中保留了Ⅰ、Ⅳ型胶原和层粘连蛋白。经鉴定,hAT-EV呈双层包膜的球形,高表达CD63、凋亡诱导因子6相互作用蛋白抗体、肿瘤易感基因101,97.9%粒径分布范围为32.67~220.20 nm,峰值为91.28 nm。共聚焦荧光显微镜和油红O染色示,hAT-EV被ADSCs摄取并诱导其成脂分化。大鼠体内实验显示,实验组新生脂肪组织湿重显著高于对照组(t=2.278,P=0.048);HE染色示,实验组脂滴�Objective To explore the possibility of constructing tissue engineered adipose by adipose tissue derived extracellular vesicles(hAT-EV)combined with decellularized adipose tissue(DAT)scaffolds,and to provide a new therapy for soft tissue defects.Methods The adipose tissue voluntarily donated by the liposuction patient was divided into two parts,one of them was decellularized and observed by HE and Masson staining and scanning electron microscope(SEM).Immunohistochemical staining and Western blot detection for collagen typeⅠandⅣand laminin were also employed.Another one was incubated with exosome-removed complete medium for 48 hours,then centrifuged to collect the medium and to obtain hAT-EV via ultracentrifugation.The morphology of hAT-EV was observed by transmission electron microscopy;the nanoparticle tracking analyzer(NanoSight)was used to analyze the size distribution;Western blot was used to analyse membrane surface protein of hAT-EV.Adipose derived stem cells(ADSCs)were cocultured with PKH26 fluorescently labeled hAT-EV,confocal fluorescence microscopy was used to observe the uptake of hAT-EV by ADSCs.Oil red O staining was used to evaluate adipogenic differentiation after hAT-EV and ADSCs cocultured for 15 days.The DAT was scissored and then injected into the bilateral backs of 8 C57 mice(6-week-old).In experimental group,0.2 mL hAT-EV was injected weekly,and 0.2 mL PBS was injected weekly in control group.After 12 weeks,the mice were sacrificed,and the new fat organisms on both sides were weighed.The amount of new fat was evaluated by HE and peri-lipoprotein immunofluorescence staining to evaluate the ability of hAT-EV to induce adipogenesis in vivo.Results After acellularization of adipose tissue,HE and Masson staining showed that DAT was mainly composed of loosely arranged collagen with no nucleus;SEM showed that no cells and cell fragments were found in DAT,and thick fibrous collagen bundles could be seen;immunohistochemical staining and Western blot detection showed that collagen typeⅠandⅣand

关 键 词：脱细胞脂肪组织支架 细胞外囊泡 脂肪干细胞 组织工程 软组织修复 小鼠 

分 类 号：R318.08[医药卫生—生物医学工程]