脂肪干细胞来源外泌体促进大鼠皮瓣移植后血管新生的研究  

Effect of adipose-derived stem cell derived exosomes on angiogenesis after skin flap transplantation in rats

作　　者：胡玄 易阳艳[1] 朱元正[1] 王朝慧[1] 吴舒[1] 张静[1] 王江文 聂佳莹 HU Xuan;YI Yangyan;ZHU Yuanzheng;WANG Zhaohui;WU Shu;ZHANG Jing;WANG Jiangwen;NIE Jiaying(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》2019年第12期1560-1565,共6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）;南昌大学研究生创新基金资助项目（CX2018206)~~

摘　　要：目的探讨脂肪干细胞来源外泌体(adipose-derived stem cell derived exosomes,ADSC-Exos)对大鼠皮瓣移植后血管新生的影响。方法取抽脂术患者自愿捐赠脂肪组织,采用酶消化法分离培养ADSCs;取第3代细胞光镜观察形态,流式细胞术鉴定细胞表面标志物,成脂诱导培养14 d后油红O染色观察。细胞鉴定为ADSCs后,采用密度梯度离心法提取ADSC-Exos;透射电镜观察形态,Western blot检测膜表面标志蛋白(CD63、TSG101),纳米颗粒跟踪分析仪测量其粒径分布。取20只雄性SD大鼠,体质量250~300 g,随机分为ADSCExos组和PBS组,每组10只。两组大鼠背部沿长轴方向制作面积为9 cm×3 cm随意皮瓣后,分别于近端、中间和远端区域注射ADSC-Exos(ADSC-Exos组)或PBS(PBS组)。术后大体观察皮瓣成活情况,第7天测算皮瓣成活率后切取皮瓣组织,HE染色观察组织形态,CD31免疫组织化学染色测定皮瓣微血管密度(mean blood vessel density,MVD)。结果光镜、流式细胞术及成脂诱导培养鉴定培养细胞为ADSCs。ADSC-Exos为边缘清晰、大小形态均匀的圆形或椭圆形膜性囊泡,高表达标志蛋白CD63和TSG101,粒径分布在30~200 nm,符合外泌体粒径范围。术后两组皮瓣远端均出现不同程度坏死表现,但ADSC-Exos组程度较PBS组轻;第7天ADSC-Exos组皮瓣成活率为64.2%±11.5%,显著大于PBS组的31.0%±6.6%(t=7.945,P=0.000);中间区域皮肤附属器官更完整,近端区域组织水肿程度轻、血管扩张更多。ADSC-Exos组MVD为(103.3±27.0)个/视野,显著多于PBS组(45.3±16.2)个/视野(t=3.190,P=0.011)。结论 ADSC-Exos可通过促进皮瓣移植后新生血管的形成,改善皮瓣血供,进而提高大鼠皮瓣成活率。Objective To investigate the effect of adipose-derived stem cell derived exosomes(ADSC-Exos) on angiogenesis after skin flap transplantation in rats. Methods ADSCs were isolated and cultured by enzymatic digestion from voluntary donated adipose tissue of patients undergoing liposuction. The 3 rd generation cells were observed under microscopy and identified by flow cytometry and oil red O staining at 14 days after induction of adipogenesis. After cells were identified as ADSCs, ADSC-Exos was extracted by density gradient centrifugation. And the morphology was observed by transmission electron microscopy, the surface marker proteins(CD63, TSG101) were detected by Western blot, and particle size distribution was measured by nanoparticle size tracking analyzer. Twenty male Sprague Dawley rats,weighing 250-300 g, were randomly divided into ADSC-Exos group and PBS group with 10 rats in each group. ADSCExos(ADSC-Exos group) and PBS(PBS group) were injected into the proximal, middle, and distal regions of the dorsal free flaps with an area of 9 cm×3 cm along the long axis in the two groups. The survival rate of the flap was measured on the 7 th day, and then the flap tissue was harvested. The tissue morphology was observed by HE staining, and mean blood vessel density(MVD) was measured by CD31 immunohistochemical staining. Results ADSCs were identified by microscopy, flow cytometry, and adipogenic induction culture. ADSC-Exos was a round or elliptical membrane vesicle with clear edge and uniform size. It has high expression of CD63 and TSG101, and its size distribution was 30-200 nm,which was in accordance with the size range of Exos. The distal necrosis of the flaps in the ADSC-Exos group was milder than that in the PBS group. On the 7 th day, the survival rate of the flaps in the ADSC-Exos group was 64.2%±11.5%, which was significantly higher than that in the PBS group(31.0%±6.6%;t=7.945, P=0.000);the skin appendages in the middle region of the flap in the ADSC-Exos group were more complete, the edema in the proxim

关 键 词：脂肪干细胞 外泌体 皮瓣移植 血管新生 大鼠 

分 类 号：R73[医药卫生—肿瘤]