预注射脂肪干细胞对皮瓣血运影响的实验观察

Study on the survival rate of random flap using pre-injection of ADSCs

目的观察预注射脂肪干细胞（ADSCs）对小鼠皮瓣血运的影响。方法体外分离培养人ADSCs，并进行流式与分化实验鉴定。将40只BALB/c小鼠按随机数字表法随机分成即刻注射组、预注射第2天组、预注射第5天组、预注射第7天组及空白对照组各8只。在小鼠背部设计1 cm×4 cm随意皮瓣，将ADSCs预先注射到预注射第2、5、7天组拟形成皮瓣的皮下组织中，于注射后第2、5、7天行皮瓣形成术，同时对即刻注射组及空白对照组行皮瓣形成术。术后第14天观察各组皮瓣的成活情况，血管性血友病因子染色法检测移植组织中微血管数量，酶联免疫吸附法检测皮瓣内血管内皮生长因子（VEGF）浓度。结果即刻注射组皮瓣成活率显著高于预注射第2、5、7天组及空白对照组[（51.5±6.3）%比（38.3±6.7）%、（21.0±2.9）%、（30.0±3.9）%及（34.5±4.2）%]，皮瓣组织中微小血管数量显著高于预注射第2、5、7天组及空白对照组[（21.0±4.3）比（17.5±3.9）、（9.9±2.6）、（13.9±2.6）及（16.1±3.3）条/低倍视野，均P〈0.01）]。皮瓣内VEGF浓度检测结果基本与皮瓣成活面积变化趋势保持一致。结论预先向拟形成皮瓣组织注射ADSCs后，在一定程度上阻碍了皮瓣的成活，并没有发挥ADSCs促血管新生的作用。

Objective To determine the effects of adipose-derived stem cells （ADSCs） on the survival rate of flaps by transplanting ADSCs into the experimental flaps pre-operatively. Methods ADSCs were isolated from fresh human fat and cultured in vitro. Their morphological characters were observed, and floweytometry and differentiation test and evaluation were conducted. A total of 40 BALB/c mice were divided into 5 groups randomly with each of 8 mice. 1 cm × 4 em random skin flap was designed on the mice back, ADSCs were injected in advance into subcutaneous tissues of 3 groups, the operation was performed on the 2^nd, 5^th and 7^th day after the ADSCs injection, meanwhile skin flap operation was conducted in the immediate injection group and control group. Survival condition of the skin flaps was observed and yon willebrand factor （vWF） staining was used to detect the number of micro-vessels in the skin flap. The enzyme-linked immuno sorbent assay （ELISA） method was used to detect the concentration of vascular endothelial growth factor （VEGF） in the skin flap. Results The immediate injection group had a significantly higher survival rate of skin flap than the pre-injeetion groups and the control group [ （51.5 ± 6.3）% vs （38.3 ±6.7）%, （21.0±2.9）%, （30.0±3.9）% and （34.5±4.2）%, all P〈0.01]. The immediate injection group had a significantly higher number of micro-vessels in the skin flap than the pre- injection groups and the control group [ （ 21.0 ± 4. 3 ） vs （ 17.5±3.9 ）, （ 9. 9 ± 2. 6）, （ 13.9 ± 2. 6 ） and （ 16. 1 ±3.3）/LPF, all P 〈0. 01 ]. The laboratory results of the concentration of VEGF in skin flap kept a consistent tendency with the flap survival rate. Conclusion Pre-injection with the ADSCs into the skin flap prevents the survival of the flap to a certain extend and does not show the function of promoting tissue angiogenesis.