摘要
目的探讨脂肪干细胞来源外泌体(adipose-derived stem cell released exosomes,ADSC-Exos)对糖尿病小鼠创面愈合的影响。方法取患者自愿捐赠脂肪组织,采用酶消化法分离培养ADSCs,并于第3代细胞上清液提取Exos(ADSC-Exos);透射电镜观察ADSC-Exos形态,Western blot检测膜表面标志性蛋白Alix、CD63,纳米颗粒跟踪分析仪检测粒径分布。取患者自愿捐赠皮肤组织,采用酶消化法分离培养成纤维细胞。将PKH26标记的ADSC-Exos与第5代成纤维细胞培养,共聚焦荧光显微镜观察其能否进入细胞,采用细胞计数试剂盒8(cell counting kit 8,CCK-8)及划痕法观察ADSC-Exos对成纤维细胞增殖及迁移的影响。取24只8周龄Balb/c雄性小鼠制备糖尿病模型后,背部制备直径8 mm全层皮肤缺损创面,实验组(n=12)及对照组(n=12)创面真皮层分别注射0.2 mL ADSC-Exos及PBS。第1、4、7、11、16、21天大体观察创面愈合情况,计算创面愈合率;第7、14、21天取材,行组织学(HE及Masson)及CD31免疫组织化学染色,观察创面结构、胶原纤维及新生血管情况。结果ADSC-Exos为边缘清晰、大小分布均匀的膜性囊泡;粒径为40~200 nm,平均102.1 nm;膜表面标志性蛋白Alix、CD63均呈阳性。ADSC-Exos与成纤维细胞复合培养观察示,ADSC-Exos可进入成纤维细胞胞内,并能促进成纤维细胞增殖及迁移。动物实验显示,实验组小鼠背部创面愈合明显快于对照组,各时间点创面愈合率差异均有统计学意义(P<0.05)。与对照组比较,实验组创面结构愈合更好,愈合前期新生微血管更多,愈合后期胶原纤维沉积更多;其中,第7、14、21天创面缺损长度,第7、14天微血管数量,第14、21天胶原纤维沉积百分比,组间差异均有统计学意义(P<0.05)。结论ADSC-Exos通过促进创面血管新生以及成纤维细胞增殖、迁移和胶原合成来促进糖尿病小鼠创面愈合。
Objective To investigate the effects of adipose-derived stem cell released exosomes(ADSC-Exos) on wound healing in diabetic mice. Methods The ADSCs were isolated from the adipose tissue donated by the patients and cultured by enzymatic digestion. The supernatant of the 3 rd generation ADSCs was used to extract Exos(ADSC-Exos).The morphology of ADSC-Exos was observed by transmission electron microscopy. The membrane-labeled proteins(Alix and CD63) were detected by Western blot, and the particle size distribution was detected by nanoparticle tracking analyzer. The fibroblasts were isolated from the skin tissue donated by the patients and cultured by enzymatic digestion.The 5 th generation fibroblasts were cultured with PKH26-labeled ADSC-Exos, and observed by confocal fluorescence microscopy. The effects of ADSC-Exos on proliferation and migration of fibroblasts were observed with cell counting kit 8(CCK-8) and scratch method. Twenty-four 8-week-old Balb/c male mice were used to prepare a diabetic model. A fullthickness skin defect of 8 mm in diameter was prepared on the back. And 0.2 mL of ADSC-Exos and PBS were injected into the dermis of the experimental group(n=12) and the control group(n=12), respectively. On the 1 st, 4 th, 7 th, 11 th,16 th, and 21 st days, the wound healing was observed and the wound healing rate was calculated. On the 7 th, 14 th, and 21 st days, the histology(HE and Masson) and CD31 immunohistochemical staining were performed to observe the wound structure, collagen fibers, and neovascularization. Results ADSC-Exos were the membranous vesicles with clear edges and uniform size;the particle size was 40-200 nm with an average of 102.1 nm;the membrane-labeled proteins(Alix and CD63) were positive. The composite culture observation showed that ADSC-Exos could enter the fibroblasts and promote the proliferation and migration of fibroblasts. Animal experiments showed that the wound healing of the experimental group was significantly faster than that of the control group, and the wound healing rate was significantly different at each time point(P<0.05). Compared with the control group, the wound healing of the experimental group was better. There were more microvessels in the early healing stage, and more deposited collagen fibers in the late healing stage. There were significant differences in the length of wound on the 7 th, 14 th, and 21 st days, the number of microvessels on the 7 th and14 th days, and the rate of deposited collagen fibers on the 14 th and 21 st days between the two groups(P<0.05).Conclusion ADSC-Exos can promote the wound healing in diabetic mice by promoting angiogenesis and proliferation and migration of fibroblasts and collagen synthesis.
作者
王江文
易阳艳
朱元正
王朝慧
吴舒
张静
胡玄
聂佳莹
WANG Jiangwen;YI Yangyan;ZHU Yuanzheng;WANG Zhaohui;WU Shu;ZHANG Jing;HU Xuan;NIE Jiaying(Department of Plastic Surgery,the Second Affiliated Hospital of Nanchang University,Nanchang Jiangxi,330006,P.R.China)
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2020年第1期124-131,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、20192BAB205056)~~
