[Effect of adipose-derived stem cell derived exosomes on angiogenesis after skin flap transplantation in rats].
[Effect of adipose-derived stem cell derived exosomes on angiogenesis after skin flap transplantation in rats].
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DOI：
10.7507/1002-1892.201904023
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发表时间：
2019-12-15
影响因子：
--
通讯作者：
Nie, Jiaying
中科院分区：
文献类型：
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作者：
Hu, Xuan;Yi, Yangyan;Nie, Jiaying
目的：探讨脂肪干细胞衍生外切体(ADSC-Exos)对大鼠皮瓣移植后血管生成的影响。方法：从自愿捐献的脂肪组织中分离培养脂肪干细胞。诱导后第14天，显微镜下观察第3代细胞，并用流式细胞仪和油红O染色进行鉴定。经鉴定为ADSCs后，密度梯度离心法提取ADSC-Exos。用透射电子显微镜观察细胞形态，Western印迹法检测细胞表面标志蛋白CD63、TSG101的表达，纳米粒子粒度跟踪分析仪测定细胞颗粒大小分布。雄性SD大鼠20只，体重250~300g，随机分为ADSC-Exos组和PBS组，每组10只。将ADSC-Exos(ADSC-Exos组)和PBS(PBS组)分别注入背侧游离皮瓣近、中、远端，长轴方向注射面积为9 cm×3 cm。术后第7天测定皮瓣成活率，取皮瓣组织。采用HE染色观察组织形态，CD31免疫组织化学染色检测平均血管密度(MVD)。结果：通过显微镜、流式细胞仪、成脂诱导培养等方法鉴定ADSC。ADSC-Exos为圆形或椭圆形的膜泡，边缘清晰，大小均匀。CD63和TSG101高表达，其大小分布在30~200 nm之间，与Exos的大小范围一致。ADSC-Exos组远端皮瓣坏死程度较PBS组轻。术后第7天，皮片成活率为64.2%±11.5%，明显高于假手术组(31.0%±6.6%；t=7.945，P=0.000)，皮片中部皮肤附属物较完整，近端水肿较轻，血管扩张较广泛。ADSC-Exos组微血管密度为(103.3±27.0)个/视野，显著高于PBS组[(45.3±16.2)个/视野；t=3.190，P=0.011]。结论：ADSC-Exos通过促进大鼠皮瓣移植后新生血管的形成，改善皮瓣血供，从而提高大鼠皮瓣存活率。
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 3rd 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. ADSC-Exos (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 7th 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 7th 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 proximal region was lighter and the vasodilation was more extensive. MVD of the ADSC-Exos group was (103.3±27.0) /field, which was significantly higher than that of the PBS group [(45.3±16.2)/field; t=3.190, P=0.011].CONCLUSION: ADSC-Exos can improve the blood supply of skin flaps by promoting the formation of neovascularization after skin flap transplantation, thereby improve the survival rate of skin flaps in rats.